Selective delivery molecules and methods of use

ABSTRACT

Disclosed herein is a selective delivery molecule comprising: (a) an acidic sequence (portion A) which is effective to inhibit or prevent the uptake into cells or tissue retention, (b) a molecular transport or retention sequence (portion B), and (c) a linker between portion A and portion B, and (d) at least one cargo moiety.

CROSS-REFERENCES

This application is the National Stage entry of International Application No. PCT/US2012/048732 filed on Jul. 27, 2012, which claims priority to U.S. Provisional Patent Application No. 61/513,287, titled “Selective Delivery Molecules and Methods of Use” and filed Jul. 29, 2011, the disclosures of which are incorporated herein by reference in their entireties.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Jan. 22, 2014, is named 39088708831.txt and is 10,684 bytes in size.

SUMMARY OF THE INVENTION

Disclosed herein, in certain embodiments, are selective delivery molecule of Formula I, having the structure: [D_(A)-c _(A)]-A-[c _(M)-M]-X-B-[c _(B)-D_(B)]  Formula I wherein,

-   -   X is a cleavable linker;     -   A is a peptide with a sequence comprising 5 to 9 acidic amino         acids;     -   B is a peptide with a sequence comprising 7 to 9 basic amino         acids;     -   c_(A), c_(B), and c_(M) each independently comprise 0-1 amino         acid;     -   M is a macromolecule; and     -   D_(A) and D_(B) are each independently selected from an imaging         agent and a therapeutic;     -   and         wherein [c_(M)-M] is bound to at any position on A or X,         [D_(A)-c_(A)] is bound to any amino acid on A, and [c_(B)-D_(B)]         is bound to any amino acid on B.         In some embodiments, A and B do not have an equal number of         acidic and basic amino acids. In some embodiments, the number of         basic amino acids in B is greater than the number of acidic         amino acids in A. In some embodiments, A is a peptide comprising         5 or 9 consecutive glutamates (SEQ ID NO: 3). In some         embodiments, B is a peptide comprising 8 or 9 consecutive         arginines (SEQ ID NO: 4). In some embodiments, A is a peptide         comprising 5 or 9 consecutive glutamates (SEQ ID NO: 3) and B is         a peptide comprising 8 or 9 consecutive arginines (SEQ ID NO:         4). In some embodiments, A is a peptide comprising 5 consecutive         glutamates (SEQ ID NO: 5) and B is a peptide comprising 8         consecutive arginines (SEQ ID NO: 6). In some embodiments,         c_(A), c_(B), and c_(M) are each independently a 0-1 amino acid.         In some embodiments, c_(A), c_(B), and c_(M) are each         independently selected from a naturally-occurring amino acid or         a non-naturally-occurring amino acid. In some embodiments,         c_(A), c_(B), and c_(M) are each independently selected from a D         amino acid, a L amino acid, an α-amino acid, a β-amino acid, or         a γ-amino acid. In some embodiments, c_(A), c_(B), and c_(M) are         each independently selected from any amino acid having a free         thiol group, any amino acid having a N-terminal amine group, and         any amino acid with a side chain capable of forming an oxime or         hydrazone bond upon reaction with a hydroxylamine or hydrazine         group. In some embodiments, c_(A), c_(B), and c_(M) are each         independently selected from D-cysteine, D-glutamate, lysine, and         para-4-acetyl L-phenylalanine. In some embodiments, c_(B) is any         amino acid having a free thiol group. In some embodiments, c_(B)         is D-cysteine. In some embodiments, c_(A) is any amino acid         having a N-terminal amine group. In some embodiments, c_(A) is         D-glutamate. In some embodiments, c_(A) is lysine. In some         embodiments, c_(M) is any amino acid with a side chain capable         of forming an oxime or hydrazone bond upon reaction with a         hydroxylamine or hydrazine group. In some embodiments, c_(M) is         para-4-acetyl L-phenylalanine. In some embodiments, X is         cleavable by a protease. In some embodiments, X is cleavable by         a matrix metalloproteinase. In some embodiments, X comprises an         amino acid sequence that is cleavable by MMP2, MMP7, MMP9, or         MMP14. In some embodiments, X comprises a peptide linkage. In         some embodiments, X comprises an amino acid sequence selected         from: PLGLAG (SEQ ID NO: 2), PLG-C(me)-AG (SEQ ID NO: 1),         RPLALWRS (SEQ ID NO: 7), ESPAYYTA (SEQ ID NO: 8), DPRSFL (SEQ ID         NO: 9), PPRSFL (SEQ ID NO: 10), RLQLKL (SEQ ID NO: 11), and         RLQLK(Ac) (SEQ ID NO: 12). In some embodiments, X comprises the         amino acid sequence PLGLAG (SEQ ID NO: 2). In some embodiments,         X comprises the amino acid sequence PLG-C(me)-AG (SEQ ID NO: 1).         In some embodiments, X comprises the amino acid sequence         RPLALWRS (SEQ ID NO: 7). In some embodiments, X comprises the         amino acid sequence DPRSFL (SEQ ID NO: 9). In some embodiments,         X comprises the amino acid sequence RLQLKL (SEQ ID NO: 11). In         some embodiments, X comprises the amino acid sequence RLQLK(Ac)         (SEQ ID NO: 12) (SEQ ID NO: 12). In some embodiments, M is         selected from a protein, a natural polymer, a synthetic polymer,         or a dendrimer. In some embodiments, M is selected from dextran,         a PEG polymer, albumin, or a combination thereof. In some         embodiments, M is a PEG. In some embodiments, M is selected from         PEG 5 kDa, PEG 12 kDa, PEG 20 kDa, PEG 30 kDa, and PEG40 kDa. In         some embodiments, D_(A) and D_(B) are a pair of acceptor and         donor fluorescent moieties that are capable of undergoing         Försters/fluorescence resonance energy transfer with the other.         In some embodiments, D_(A) and D_(B) are Cy5 and Cy7. In some         embodiments, D_(A) and D_(B) are Cy5 and IRDye750. In some         embodiments, D_(A) and D_(B) are Cy5 and IRDye800. In some         embodiments, D_(A) and D_(B) are Cy5 and ICG. In some         embodiments, D_(A) and D_(B) are a fluorescent moiety and a         fluorescence-quenching moiety. In some embodiments, the molecule         of Formula I is: SDM-14, SDM-15, SDM-23, SDM-24, SDM-25, SDM-26,         SDM-27, SDM-32, or SDM-35.

Disclosed herein, in certain embodiments, are selective delivery molecules of Formula I, having the structure: [D_(A)-c _(A)]-A-[c _(M)-M]-X-B-[c _(B)-D_(B)]  Formula I wherein,

-   -   X is a cleavable linker;     -   A is a peptide with a sequence comprising 5 to 9 acidic amino         acids;     -   B is a peptide with a sequence comprising 7 to 9 basic amino         acids;     -   c_(A), c_(B), and c_(M) each independently comprise 0-1 amino         acid;     -   M is a polyethylene glycol (PEG) polymer; and     -   D_(A) and D_(B) are each independently an imaging agent; and         wherein [c_(M)-M] is bound to at any position on A or X,         [D_(A)-c_(A)] is bound to any amino acid on A, and [c_(B)-D_(B)]         is bound to any amino acid on B.         In some embodiments, A and B do not have an equal number of         acidic and basic amino acids. In some embodiments, the number of         basic amino acids in B is greater than the number of acidic         amino acids in A. In some embodiments, A is a peptide comprising         5 or 9 consecutive glutamates (SEQ ID NO: 3). In some         embodiments, B is a peptide comprising 8 or 9 consecutive         arginines (SEQ ID NO: 4). In some embodiments, A is a peptide         comprising 5 or 9 consecutive glutamates (SEQ ID NO: 3) and B is         a peptide comprising 8 or 9 consecutive arginines (SEQ ID NO:         4). In some embodiments, A is a peptide comprising 5 consecutive         glutamates (SEQ ID NO: 5) and B is a peptide comprising 8         consecutive arginines (SEQ ID NO: 6). In some embodiments,         c_(A), c_(B), and c_(M) are each independently a 0-1 amino acid.         In some embodiments, c_(A), c_(B), and c_(M) are each         independently selected from a naturally-occurring amino acid or         a non-naturally-occurring amino acid. In some embodiments,         c_(A), c_(B), and c_(M) are each independently selected from a D         amino acid, a L amino acid, an α-amino acid, a β-amino acid, or         a γ-amino acid. In some embodiments, c_(A), c_(B), and c_(M) are         each independently selected from any amino acid having a free         thiol group, any amino acid having a N-terminal amine group, and         any amino acid with a side chain capable of forming an oxime or         hydrazone bond upon reaction with a hydroxylamine or hydrazine         group. In some embodiments, c_(A), c_(B), and c_(M) are each         independently selected from D-cysteine, D-glutamate, lysine, and         para-4-acetyl L-phenylalanine. In some embodiments, c_(B) is any         amino acid having a free thiol group. In some embodiments, c_(B)         is D-cysteine. In some embodiments, c_(A) is any amino acid         having a N-terminal amine group. In some embodiments, c_(A) is         D-glutamate. In some embodiments, c_(A) is lysine. In some         embodiments, c_(M) is any amino acid with a side chain capable         of forming an oxime or hydrazone bond upon reaction with a         hydroxylamine or hydrazine group. In some embodiments, c_(M) is         para-4-acetyl L-phenylalanine. In some embodiments, X is         cleavable by a protease. In some embodiments, X is cleavable by         a matrix metalloproteinase. In some embodiments, X comprises an         amino acid sequence that is cleavable by MMP2, MMP7, MMP9, or         MMP14. In some embodiments, X comprises a peptide linkage. In         some embodiments, X comprises an amino acid sequence selected         from: PLGLAG (SEQ ID NO: 2), PLG-C(me)-AG (SEQ ID NO: 1),         RPLALWRS (SEQ ID NO: 7), ESPAYYTA (SEQ ID NO: 8), DPRSFL (SEQ ID         NO: 9), PPRSFL (SEQ ID NO: 10), RLQLKL (SEQ ID NO: 11), and         RLQLK(Ac) (SEQ ID NO: 12). In some embodiments, X comprises the         amino acid sequence PLGLAG (SEQ ID NO: 2). In some embodiments,         X comprises the amino acid sequence PLG-C(me)-AG (SEQ ID NO: 1).         In some embodiments, X comprises the amino acid sequence         RPLALWRS (SEQ ID NO: 7). In some embodiments, X comprises the         amino acid sequence DPRSFL (SEQ ID NO: 9). In some embodiments,         X comprises the amino acid sequence PPRSFL (SEQ ID NO: 10). In         some embodiments, X comprises the amino acid sequence RLQLKL         (SEQ ID NO: 11). In some embodiments, X comprises the amino acid         sequence RLQLK(Ac) (SEQ ID NO: 12). In some embodiments, D_(A)         and D_(B) are a pair of acceptor and donor fluorescent moieties         that are capable of undergoing Försters/fluorescence resonance         energy transfer with the other. In some embodiments, D_(A) and         D_(B) are Cy5 and Cy7. In some embodiments, D_(A) and D_(B) are         Cy5 and IRDye750. In some embodiments, D_(A) and D_(B) are Cy5         and IRDye800. In some embodiments, D_(A) and D_(B) are Cy5 and         ICG. In some embodiments, D_(A) and D_(B) are a fluorescent         moiety and a fluorescence-quenching moiety. In some embodiments,         the molecule of Formula I is: SDM-14, SDM-15, SDM-23, SDM-24,         SDM-25, SDM-26, SDM-27, SDM-32; or SDM-35.

Disclosed herein, in certain embodiments, are molecules of Formula II, having the structure: A₁-X₁-B₁;  Formula II wherein,

-   -   X₁ is a cleavable linker;     -   A₁ is a peptide with a sequence comprising 5 to 9 acidic amino         acids and having a first reactive amino acid moiety c_(A);     -   B₁ is a peptide with a sequence comprising 7 to 9 basic amino         acids and having a second reactive amino acid moiety c_(B); and     -   A₁-X₁-B₁ has a third reactive amino acid moiety c_(M) on A₁ or         X₁; and         wherein c_(A) is capable of reacting with a first cargo moiety         comprising D_(A), c_(B) is capable of reacting with a second         cargo moiety comprising D_(B), and c_(M) is capable of reacting         with a macromolecular carrier comprising M to form a molecule of         Formula I.         In some embodiments, c_(A), c_(B), and c_(M) are each         independently a 0-1 amino acid. In some embodiments, the c_(A),         c_(B), and c_(M) have functional groups that are orthogonally         reactive. In some embodiments, c_(A), c_(B), and c_(M) are each         independently selected from a naturally-occurring amino acid or         a non-naturally-occurring amino acid. In some embodiments,         c_(A), c_(B), and c_(M) are each independently selected from a D         amino acid, a L amino acid, an α-amino acid, a β-amino acid, or         a γ-amino acid. In some embodiments, c_(A), c_(B), and c_(M) are         each independently selected from any amino acid having a free         thiol group, any amino acid having a N-terminal amine group, and         any amino acid with a side chain capable of forming an oxime or         hydrazone bond upon reaction with a hydroxylamine or hydrazine         group. In some embodiments, c_(A), c_(B), and c_(M) are each         independently selected from D-cysteine, D-glutamate, lysine, and         para-4-acetyl L-phenylalanine. In some embodiments, c_(B) is any         amino acid having a free thiol group. In some embodiments, c_(B)         is D-cysteine. In some embodiments, c_(A) is any amino acid         having a N-terminal amine group. In some embodiments, c_(A) is         D-glutamate. In some embodiments, c_(A) is lysine. In some         embodiments, c_(M) is any amino acid with a side chain capable         of forming an oxime or hydrazone bond upon reaction with a         hydroxylamine or hydrazine group. In some embodiments, c_(M) is         para-4-acetyl L-phenylalanine.

Disclosed herein, in certain embodiments, are tissue samples comprising a molecule of Formula I: [D_(A)-c _(A)]-A-[c _(M)-M]-X-B-[c _(B)-D_(B)];  Formula I wherein,

-   -   X is a cleavable linker;     -   A is a peptide with a sequence comprising 5 to 9 acidic amino         acids;     -   B is a peptide with a sequence comprising 7 to 9 basic amino         acids;     -   c_(A), c_(B), and c_(M) each independently comprise 0-1 amino         acid;     -   M is a polyethylene glycol (PEG) polymer; and     -   D_(A) and D_(B) are each independently an imaging agent; and         wherein [c_(M)-M] is bound at any position on A or X,         [D_(A)-c_(A)] is bound to any amino acid on A, and [c_(B)-D_(B)]         is bound to any amino acid on B.         In some embodiments, the tissue sample is a pathology slide or         section. In some embodiments, the tissue sample is cancerous. In         some embodiments, the cancerous tissue is: breast cancer tissue,         colon cancer tissue, squamous cell carcinoma tissue, prostate         cancer tissue, melanoma tissue, or thyroid cancer tissue. In         some embodiments, the cancerous tissue is breast cancer tissue.         In some embodiments, the cancerous tissue is colon cancer         tissue. In some embodiments, A and B do not have an equal number         of acidic and basic amino acids. In some embodiments, the number         of basic amino acids in B is greater than the number of acidic         amino acids in A. In some embodiments, A is a peptide comprising         5 or 9 consecutive glutamates (SEQ ID NO: 3). In some         embodiments, B is a peptide comprising 8 or 9 consecutive         arginines (SEQ ID NO: 4). In some embodiments, A is a peptide         comprising 5 or 9 consecutive glutamates (SEQ ID NO: 3) and B is         a peptide comprising 8 or 9 consecutive arginines (SEQ ID NO:         4). In some embodiments, A is a peptide comprising 5 consecutive         glutamates (SEQ ID NO: 5) and B is a peptide comprising 8         consecutive arginines (SEQ ID NO: 6). In some embodiments,         c_(A), c_(B), and c_(M) are each independently a 0-1 amino acid.         In some embodiments, c_(A), c_(B), and c_(M) are each         independently selected from a naturally-occurring amino acid or         a non-naturally-occurring amino acid. In some embodiments,         c_(A), c_(B), and c_(M) are each independently selected from a D         amino acid, a L amino acid, an α-amino acid, a β-amino acid, or         a γ-amino acid. In some embodiments, c_(A), c_(B), and c_(M) are         each independently selected from any amino acid having a free         thiol group, any amino acid having a N-terminal amine group, and         any amino acid with a side chain capable of forming an oxime or         hydrazone bond upon reaction with a hydroxylamine or hydrazine         group. In some embodiments, c_(A), c_(B), and c_(M) are each         independently selected from D-cysteine, D-glutamate, lysine, and         para-4-acetyl L-phenylalanine. In some embodiments, c_(B) is any         amino acid having a free thiol group. In some embodiments, c_(B)         is D-cysteine. In some embodiments, c_(A) is any amino acid         having a N-terminal amine group. In some embodiments, c_(A) is         D-glutamate. In some embodiments, c_(A) is lysine. In some         embodiments, c_(M) is any amino acid with a side chain capable         of forming an oxime or hydrazone bond upon reaction with a         hydroxylamine or hydrazine group. In some embodiments, c_(M) is         para-4-acetyl L-phenylalanine. In some embodiments, X is         cleavable by a protease. In some embodiments, X is cleavable by         a matrix metalloproteinase. In some embodiments, X comprises an         amino acid sequence that is cleavable by MMP2, MMP7, MMP9, or         MMP14. In some embodiments, X comprises a peptide linkage. In         some embodiments, X comprises an amino acid sequence selected         from: PLGLAG (SEQ ID NO: 2), PLG-C(me)-AG (SEQ ID NO: 1),         RPLALWRS (SEQ ID NO: 7), ESPAYYTA (SEQ ID NO: 8), DPRSFL (SEQ ID         NO: 9), PPRSFL (SEQ ID NO: 10), RLQLKL (SEQ ID NO: 11), and         RLQLK(Ac) (SEQ ID NO: 12). In some embodiments, X comprises the         amino acid sequence PLGLAG (SEQ ID NO: 2). In some embodiments,         X comprises the amino acid sequence PLG-C(me)-AG (SEQ ID NO: 1).         In some embodiments, X comprises the amino acid sequence         RPLALWRS (SEQ ID NO: 7). In some embodiments, X comprises the         amino acid sequence DPRSFL (SEQ ID NO: 9). In some embodiments,         X comprises the amino acid sequence PPRSFL (SEQ ID NO: 10). In         some embodiments, X comprises the amino acid sequence RLQLKL         (SEQ ID NO: 11). In some embodiments, X comprises the amino acid         sequence RLQLK(Ac) (SEQ ID NO: 12). In some embodiments, D_(A)         and D_(B) are a pair of acceptor and donor fluorescent moieties         that are capable of undergoing Försters/fluorescence resonance         energy transfer with the other. In some embodiments, D_(A) and         D_(B) are Cy5 and Cy7. In some embodiments, D_(A) and D_(B) are         Cy5 and IRDye750. In some embodiments, D_(A) and D_(B) are Cy5         and IRDye800. In some embodiments, D_(A) and D_(B) are Cy5 and         ICG. In some embodiments, D_(A) and D_(B) are a fluorescent         moiety and a fluorescence-quenching moiety. In some embodiments,         the molecule of Formula I is: SDM-14, SDM-15, SDM-23, SDM-24,         SDM-25, SDM-26, SDM-27, SDM-32, and SDM-35.

Disclosed herein, in certain embodiments, are methods of delivering a pair of imaging agents to a tissue of interest, comprising contacting the tissue of interest with a molecule of Formula I: [D_(A)-c _(A)]-A-[c _(M)-M]-X-B-[c _(B)-D_(B)];  Formula I wherein,

-   -   X is a cleavable linker;     -   A is a peptide with a sequence comprising 5 to 9 acidic amino         acids;     -   B is a peptide with a sequence comprising 7 to 9 basic amino         acids;     -   c_(A), c_(B), and c_(M) each independently comprise 0-1 amino         acid;     -   M is a polyethylene glycol (PEG) polymer; and     -   D_(A) and D_(B) are each independently an imaging agent; and         wherein [c_(M)-M] is bound to at any position on A or X,         [D_(A)-c_(A)] is bound to any amino acid on A, and [c_(B)-D_(B)]         is bound to any amino acid on B.         In some embodiments, the tissue of interest is cancerous. In         some embodiments, the cancerous tissue is: breast cancer tissue,         colorectal cancer tissue, squamous cell carcinoma tissue,         prostate cancer tissue, melanoma tissue, and thyroid cancer         tissue. In some embodiments, the cancerous tissue is breast         cancer tissue. In some embodiments, the cancerous tissue is         colon cancer tissue. In some embodiments, A and B do not have an         equal number of acidic and basic amino acids. In some         embodiments, the number of basic amino acids in B is greater         than the number of acidic amino acids in A. In some embodiments,         A is a peptide comprising 5 or 9 consecutive glutamates (SEQ ID         NO: 3). In some embodiments, B is a peptide comprising 8 or 9         consecutive arginines (SEQ ID NO: 4). In some embodiments, A is         a peptide comprising 5 or 9 consecutive glutamates (SEQ ID         NO: 3) and B is a peptide comprising 8 or 9 consecutive         arginines (SEQ ID NO: 4). In some embodiments, A is a peptide         comprising 5 consecutive glutamates (SEQ ID NO: 5) and B is a         peptide comprising 8 consecutive arginines (SEQ ID NO: 6). In         some embodiments, c_(A), c_(B), and c_(M) are each independently         a 0-1 amino acid. In some embodiments, c_(A), c_(B), and c_(M)         are each independently selected from a naturally-occurring amino         acid or a non-naturally-occurring amino acid. In some         embodiments, c_(A), c_(B), and c_(M) are each independently         selected from a D amino acid, a L amino acid, an α-amino acid, a         β-amino acid, or a γ-amino acid. In some embodiments, c_(A),         c_(B), and c_(M) are each independently selected from any amino         acid having a free thiol group, any amino acid having a         N-terminal amine group, and any amino acid with a side chain         capable of forming an oxime or hydrazone bond upon reaction with         a hydroxylamine or hydrazine group. In some embodiments, c_(A),         c_(B), and c_(M) are each independently selected from         D-cysteine, D-glutamate, lysine, and para-4-acetyl         L-phenylalanine. In some embodiments, c_(B) is any amino acid         having a free thiol group. In some embodiments, c_(B) is         D-cysteine. In some embodiments, c_(A) is any amino acid having         a N-terminal amine group. In some embodiments, c_(A) is         D-glutamate. In some embodiments, c_(A) is lysine. In some         embodiments, c_(M) is any amino acid with a side chain capable         of forming an oxime or hydrazone bond upon reaction with a         hydroxylamine or hydrazine group. In some embodiments, c_(M) is         para-4-acetyl L-phenylalanine. In some embodiments, X is         cleavable by a protease. In some embodiments, X is cleavable by         a matrix metalloproteinase. In some embodiments, X comprises an         amino acid sequence that is cleavable by MMP2, MMP7, MMP9, or         MMP14. In some embodiments, X comprises a peptide linkage. In         some embodiments, X comprises an amino acid sequence selected         from: PLGLAG (SEQ ID NO: 2), PLG-C(me)-AG (SEQ ID NO: 1),         RPLALWRS (SEQ ID NO: 7), ESPAYYTA (SEQ ID NO: 8), DPRSFL (SEQ ID         NO: 9), PPRSFL (SEQ ID NO: 10), RLQLKL (SEQ ID NO: 11), and         RLQLK(Ac) (SEQ ID NO: 12). In some embodiments, X comprises the         amino acid sequence PLGLAG (SEQ ID NO: 2). In some embodiments,         X comprises the amino acid sequence PLG-C(me)-AG (SEQ ID NO: 1).         In some embodiments, X comprises the amino acid sequence         RPLALWRS (SEQ ID NO: 7). In some embodiments, X comprises the         amino acid sequence DPRSFL (SEQ ID NO: 9). In some embodiments,         X comprises the amino acid sequence PPRSFL (SEQ ID NO: 10). In         some embodiments, X comprises the amino acid sequence RLQLKL         (SEQ ID NO: 11). In some embodiments, X comprises the amino acid         sequence RLQLK(Ac) (SEQ ID NO: 12). In some embodiments, D_(A)         and D_(B) are a pair of acceptor and donor fluorescent moieties         that are capable of undergoing Försters/fluorescence resonance         energy transfer with the other. In some embodiments, D_(A) and         D_(B) are Cy5 and Cy7. In some embodiments, D_(A) and D_(B) are         Cy5 and IRDye750. In some embodiments, D_(A) and D_(B) are Cy5         and IRDye800. In some embodiments, D_(A) and D_(B) are Cy5 and         ICG. In some embodiments, D_(A) and D_(B) are a fluorescent         moiety and a fluorescence-quenching moiety. In some embodiments,         the molecule of Formula I is: SDM-14, SDM-15, SDM-23, SDM-24,         SDM-25, SDM-26, SDM-27, SDM-32, and SDM-35.

Disclosed herein, in certain embodiments, are methods of visualizing a tissue of interest in an individual in need thereof, comprising:

(a) administering to the individual a molecule of Formula I that localizes to the tissue of interest in the individual, [D_(A)-c _(A)]-A-[c _(M)-M]-X-B-[c _(B)-D_(B)];  Formula I wherein,

-   -   X is a cleavable linker;     -   A is a peptide with a sequence comprising 5 to 9 acidic amino         acids;     -   B is a peptide with a sequence comprising 7 to 9 basic amino         acids;     -   c_(A), c_(B), and c_(M) each independently comprise 0-1 amino         acid;     -   M is a polyethylene glycol (PEG) polymer; and     -   D_(A) and D_(B) are each independently an imaging agent; and     -   wherein [c_(M)-M] is bound to at any position on A or X,         [D_(A)-c_(A)] is bound to any amino acid on A, and [c_(B)-D_(B)]         is bound to any amino acid on B; and         (b) visualizing at least one of the imaging agents.         In some embodiments, the tissue is cancerous. In some         embodiments, the cancerous tissue is: breast cancer tissue,         colorectal cancer tissue, squamous cell carcinoma tissue,         prostate cancer tissue, melanoma tissue, or thyroid cancer         tissue. In some embodiments, the cancerous cell or tissue is         breast cancer tissue. In some embodiments, the cancerous cell or         tissue is colon cancer tissue. In some embodiments, the method         further comprises surgically removing the tissue of interest         from the individual. In some embodiments, the surgical margin         surrounding the tissue of interest is decreased. In some         embodiments, the method further comprises preparing a tissue         sample from the removed cell or tissue of interest. In some         embodiments, the method further comprises staging the cancerous         tissue. In some embodiments, A and B do not have an equal number         of acidic and basic amino acids. In some embodiments, the number         of basic amino acids in B is greater than the number of acidic         amino acids in A. In some embodiments, A is a peptide comprising         5 or 9 consecutive glutamates (SEQ ID NO: 3). In some         embodiments, B is a peptide comprising 8 or 9 consecutive         arginines (SEQ ID NO: 4). In some embodiments, A is a peptide         comprising 5 or 9 consecutive glutamates (SEQ ID NO: 3) and B is         a peptide comprising 8 or 9 consecutive arginines (SEQ ID NO:         4). In some embodiments, A is a peptide comprising 5 consecutive         glutamates (SEQ ID NO: 5) and B is a peptide comprising 8         consecutive arginines (SEQ ID NO: 6). In some embodiments,         c_(A), c_(B), and c_(M) are each independently a 0-1 amino acid.         In some embodiments, c_(A), c_(B), and c_(M) are each         independently selected from a naturally-occurring amino acid or         a non-naturally-occurring amino acid. In some embodiments,         c_(A), c_(B), and c_(M) are each independently selected from a D         amino acid, a L amino acid, an α-amino acid, a β-amino acid, or         a γ-amino acid. In some embodiments, c_(A), c_(B), and c_(M) are         each independently selected from any amino acid having a free         thiol group, any amino acid having a N-terminal amine group, and         any amino acid with a side chain capable of forming an oxime or         hydrazone bond upon reaction with a hydroxylamine or hydrazine         group. In some embodiments, c_(A), c_(B), and c_(M) are each         independently selected from D-cysteine, D-glutamate, lysine, and         para-4-acetyl L-phenylalanine. In some embodiments, c_(B) is any         amino acid having a free thiol group. In some embodiments, c_(B)         is D-cysteine. In some embodiments, c_(A) is any amino acid         having a N-terminal amine group. In some embodiments, c_(A) is         D-glutamate. In some embodiments, c_(A) is lysine. In some         embodiments, c_(M) is any amino acid with a side chain capable         of forming an oxime or hydrazone bond upon reaction with a         hydroxylamine or hydrazine group. In some embodiments, c_(M) is         para-4-acetyl L-phenylalanine. In some embodiments, X is         cleavable by a protease. In some embodiments, X is cleavable by         a matrix metalloproteinase. In some embodiments, X comprises an         amino acid sequence that is cleavable by MMP2, MMP7, MMP9, or         MMP14. In some embodiments, X comprises a peptide linkage. In         some embodiments, X comprises an amino acid sequence selected         from: PLGLAG (SEQ ID NO: 2), PLG-C(me)-AG (SEQ ID NO: 1),         RPLALWRS (SEQ ID NO: 7), ESPAYYTA (SEQ ID NO: 8), DPRSFL (SEQ ID         NO: 9), PPRSFL (SEQ ID NO: 10), RLQLKL (SEQ ID NO: 11), and         RLQLK(Ac) (SEQ ID NO: 12). In some embodiments, X comprises the         amino acid sequence PLGLAG (SEQ ID NO: 2). In some embodiments,         X comprises the amino acid sequence PLG-C(me)-AG (SEQ ID NO: 1).         In some embodiments, X comprises the amino acid sequence         RPLALWRS (SEQ ID NO: 7). In some embodiments, X comprises the         amino acid sequence DPRSFL (SEQ ID NO: 9). In some embodiments,         X comprises the amino acid sequence PPRSFL (SEQ ID NO: 10). In         some embodiments, X comprises the amino acid sequence RLQLKL         (SEQ ID NO: 11). In some embodiments, X comprises the amino acid         sequence RLQLK(Ac) (SEQ ID NO: 12). In some embodiments, D_(A)         and D_(B) are a pair of acceptor and donor fluorescent moieties         that are capable of undergoing Försters/fluorescence resonance         energy transfer with the other. In some embodiments, D_(A) and         D_(B) are Cy5 and Cy7. In some embodiments, D_(A) and D_(B) are         Cy5 and IRDye750. In some embodiments, D_(A) and D_(B) are Cy5         and IRDye800. In some embodiments, D_(A) and D_(B) are Cy5 and         ICG. In some embodiments, the method further comprises         visualizing Försters/fluorescence resonance energy transfer         between D_(A) and D_(B). In some embodiments, D_(A) and D_(B)         are a fluorescent moiety and a fluorescence-quenching moiety. In         some embodiments, the molecule is chosen from: SDM-14, SDM-15,         SDM-23, SDM-24, SDM-25, SDM-26, SDM-27, SDM-32, and SDM-35.

Disclosed herein, in certain embodiments, are selective delivery molecules of Formula I, having the structure: [D_(A)-c _(A)]-A-[c _(M)-M]-X-B-[c _(B)-D_(B)]  Formula I wherein,

-   -   X is a peptide linker cleavable by a matrix metalloproteinase;     -   A is a peptide with a sequence comprising 5 or 9 consecutive         glutamates (SEQ ID NO: 3);     -   B is a peptide with a sequence comprising 8 or 9 consecutive         arginines (SEQ ID NO: 4);     -   c_(A), c_(B), and c_(M) each independently comprise 0-1 amino         acid;     -   M is a polyethylene glycol (PEG) polymer; and     -   D_(A) and D_(B) are a pair of acceptor and donor fluorescent         moieties that are capable of undergoing Försters/fluorescence         resonance energy transfer with the other; and         wherein [c_(M)-M] is bound to at any position on A or X,         [D_(A)-c_(A)] is bound to any amino acid on A, and [c_(B)-D_(B)]         is bound to any amino acid on B.         In some embodiments, A and B do not have an equal number of         acidic and basic amino acids. In some embodiments, c_(A), c_(B),         and c_(M) are each independently a 0-1 amino acid. In some         embodiments, c_(A), c_(B), and c_(M) are each independently         selected from any amino acid having a free thiol group, any         amino acid having a N-terminal amine group, and any amino acid         with a side chain capable of forming an oxime or hydrazone bond         upon reaction with a hydroxylamine or hydrazine group. In some         embodiments, c_(A), c_(B), and c_(M) are each independently         selected from D-cysteine, D-glutamate, lysine, and para-4-acetyl         L-phenylalanine. In some embodiments, c_(B) is any amino acid         having a free thiol group. In some embodiments, c_(B) is         D-cysteine. In some embodiments, c_(A) is any amino acid having         a N-terminal amine group. In some embodiments, c_(A) is         D-glutamate. In some embodiments, c_(M) is any amino acid with a         side chain capable of forming an oxime or hydrazone bond upon         reaction with a hydroxylamine or hydrazine group. In some         embodiments, c_(M) is para-4-acetyl L-phenylalanine. In some         embodiments, X comprises the amino acid sequence PLGLAG (SEQ ID         NO: 2). In some embodiments, X comprises the amino acid sequence         PLG-C(me)-AG (SEQ ID NO: 1). In some embodiments, X comprises         the amino acid sequence RPLALWRS (SEQ ID NO: 7). In some         embodiments, D_(A) and D_(B) are Cy5 and Cy7. In some         embodiments, D_(A) and D_(B) are Cy5 and IRDye750. In some         embodiments, D_(A) and D_(B) are Cy5 and IRDye800. In some         embodiments, D_(A) and D_(B) are Cy5 and ICG.

Disclosed herein, in certain embodiments, are selective delivery molecules of Formula I, having the structure: [D_(A)-c _(A)]-A-[c _(M)-M]-X-B-[c _(B)-D_(B)]  Formula I wherein,

-   -   X is a peptide linker cleavable by a matrix metalloproteinase;     -   A is a peptide with a sequence comprising 5 consecutive         glutamates (SEQ ID NO: 5);     -   B is a peptide with a sequence comprising 8 consecutive         arginines (SEQ ID NO: 6);     -   c_(A), c_(B), and c_(M) each independently comprise 0-1 amino         acid;     -   M is a polyethylene glycol (PEG) polymer; and     -   D_(A) and D_(B) are a pair of acceptor and donor fluorescent         moieties that are capable of undergoing Försters/fluorescence         resonance energy transfer with the other; and         wherein [c_(M)-M] is bound to at any position on A or X,         [D_(A)-c_(A)] is bound to any amino acid on A, and [c_(B)-D_(B)]         is bound to any amino acid on B.         In some embodiments, c_(A), c_(B), and c_(M) are each         independently selected from any amino acid having a free thiol         group, any amino acid having a N-terminal amine group, and any         amino acid with a side chain capable of forming an oxime or         hydrazone bond upon reaction with a hydroxylamine or hydrazine         group. In some embodiments, c_(A), c_(B), and c_(M) are each         independently a 0-1 amino acid. In some embodiments, c_(A),         c_(B), and c_(M) are each independently selected from         D-cysteine, D-glutamate, lysine, and para-4-acetyl         L-phenylalanine. In some embodiments, c_(B) is any amino acid         having a free thiol group. In some embodiments, c_(B) is         D-cysteine. In some embodiments, c_(A) is any amino acid having         a N-terminal amine group. In some embodiments, c_(A) is         D-glutamate. In some embodiments, c_(M) is any amino acid with a         side chain capable of forming an oxime or hydrazone bond upon         reaction with a hydroxylamine or hydrazine group. In some         embodiments, c_(M) is para-4-acetyl L-phenylalanine. In some         embodiments, X comprises the amino acid sequence PLGLAG (SEQ ID         NO: 2). In some embodiments, X comprises the amino acid sequence         PLG-C(me)-AG (SEQ ID NO: 1). In some embodiments, X comprises         the amino acid sequence RPLALWRS (SEQ ID NO: 7). In some         embodiments, D_(A) and D_(B) are Cy5 and Cy7. In some         embodiments, D_(A) and D_(B) are Cy5 and IRDye750. In some         embodiments, D_(A) and D_(B) are Cy5 and IRDye800. In some         embodiments, D_(A) and D_(B) are Cy5 and ICG.

Disclosed herein, in certain embodiments, are selective delivery molecules of Formula I, having the structure: [D_(A)-c _(A)]-A-[c _(M)-M]-X-B-[c _(B)-D_(B)]  Formula I wherein,

-   -   X is a peptide linker cleavable by a matrix metalloproteinase;     -   A is a peptide with a sequence comprising 9 consecutive         glutamates (SEQ ID NO: 13);     -   B is a peptide with a sequence comprising 9 consecutive         arginines (SEQ ID NO: 14); c_(A), c_(B), and c_(M) each         independently comprise 0-1 amino acid;     -   M is a polyethylene glycol (PEG) polymer; and     -   D_(A) and D_(B) are a pair of acceptor and donor fluorescent         moieties that are capable of undergoing Försters/fluorescence         resonance energy transfer with the other; and         wherein [c_(M)-M] is bound to at any position on A or X,         [D_(A)-c_(A)] is bound to any amino acid on A, and [c_(B)-D_(B)]         is bound to any amino acid on B.         In some embodiments, c_(A), c_(B), and c_(M) are each         independently selected from any amino acid having a free thiol         group, any amino acid having a N-terminal amine group, and any         amino acid with a side chain capable of forming an oxime or         hydrazone bond upon reaction with a hydroxylamine or hydrazine         group. In some embodiments, c_(A), c_(B), and c_(M) are each         independently a 0-1 amino acid. In some embodiments, c_(A),         c_(B), and c_(M) are each independently selected from         D-cysteine, D-glutamate, lysine, and para-4-acetyl         L-phenylalanine. In some embodiments, c_(B) is any amino acid         having a free thiol group. In some embodiments, c_(B) is         D-cysteine. In some embodiments, c_(A) is any amino acid having         a N-terminal amine group. In some embodiments, c_(A) is         D-glutamate. In some embodiments, c_(M) is any amino acid with a         side chain capable of forming an oxime or hydrazone bond upon         reaction with a hydroxylamine or hydrazine group. In some         embodiments, c_(M) is para-4-acetyl L-phenylalanine. In some         embodiments, X comprises the amino acid sequence PLGLAG (SEQ ID         NO: 2). In some embodiments, X comprises the amino acid sequence         PLG-C(me)-AG (SEQ ID NO: 1). In some embodiments, X comprises         the amino acid sequence RPLALWRS (SEQ ID NO: 7). In some         embodiments, D_(A) and D_(B) are Cy5 and Cy7. In some         embodiments, D_(A) and D_(B) are Cy5 and IRDye750. In some         embodiments, D_(A) and D_(B) are Cy5 and IRDye800. In some         embodiments, D_(A) and D_(B) are Cy5 and ICG.

Disclosed herein, in certain embodiments, are selective delivery molecules according to SDM-14.

Disclosed herein, in certain embodiments, are selective delivery molecules according to SDM-15.

Disclosed herein, in certain embodiments, are selective delivery molecules according to SDM-23.

Disclosed herein, in certain embodiments, are selective delivery molecules according to SDM-24.

Disclosed herein, in certain embodiments, are selective delivery molecules according to SDM-25.

Disclosed herein, in certain embodiments, are selective delivery molecules according to SDM-26.

Disclosed herein, in certain embodiments, are selective delivery molecules according to SDM-27.

Disclosed herein, in certain embodiments, are selective delivery molecules according to SDM-32.

Disclosed herein, in certain embodiments, are selective delivery molecules according to SDM-35.

Disclosed herein, in certain embodiments, are peptides according to Peptide P-3.

FIGURES

FIG. 1 exemplifies the effects of a selective delivery molecule (SDM) by showing whole mouse fluorescence images of 3 different mice injected with SDM-6. The images were taken 2 hours after injection. The tumor and contra-lateral tissue used to calculate the contrast are indicated on the right hand mouse. The mean contrast for the three mice is 1.1.

FIG. 2 exemplifies ratiometric fluorescence changes of selective delivery molecules. In this figure, SDM-9 was cleaved with with 1 nM MMP-2 enzyme. The increase of donor (left panel) and decrease in acceptor (right panel) fluorescence is indicative of decreased FRET after peptide cleavage.

FIG. 3 exemplifies fluorescence enhancement of selective delivery molecules after protease cleavage. SDM-10 was cleaved with 1 nM MMP-9 enzyme in buffered saline. The Cy5 fluorescence increases >100 fold after peptide cleavage because the quencher dyes is no longer intramolecularly attached to Cy5 and it can no longer efficiently quench Cy5.

FIG. 4 exemplifies fluorescence enhancement of selective delivery molecule SDM-10 upon cleavage by tumor homogenates. Selective delivery molecule 10 (SDM-10) was cleaved with HT-1080 tumor homogenates. 1 nM MMP-9 or 10 uL tumor tissue homogenates (TH2 and TH3) were mixed with 1 uM compound 13 in 100 uL buffer for 24 h at 37° C. GM6001 is a general broad spectrum inhibitor of MMPs. The control lane contains SDM-6 which is highly fluorescent in the intact, uncleaved form which runs at the top of the gel. Uncleaved SDM-10 is nonfluorescent due to efficient quenching (second column from left). After cleavage by MMP-9 the fragment containing the fluorophore is dequenched (becoming highly fluorescent) and runs near the bottom of the gel. As demonstrated in the gel, tumor homogenates also cleave SDM-10 to generate the highly fluorescent product. This reaction is blocked by the MMP inhibitor indicating that the cleavage is due to tumor associated MMPs.

FIG. 5 exemplifies the bio-distribution of 3 fluorescent compounds 6 hours after IV tail vein administration of 2.9 nmol of each compound. SDM-6 has 5-fold higher tissue distribution into tumor compared to SDM-1 and SDM-2. Selective delivery molecules 1 and 2 have equal numbers of glutamates and arginines giving them a net neutral core while SDM-6 has a net 3+ charge due to more positively charged arginines.

FIG. 6 exemplifies application of emission ratio imaging of FRET to determine the presence of cancer in mouse lymph nodes. An emission ratio image was generated using equation 2 where Exp1=0.7 sec, Exp2=4.1 sec and k=20. The right hand panel show the ratio image which show high contrast between the metastatic lymph node (very large node indicated with lower left dark arrow) and the non-metastatic nodes (other arrows). The higher ratio is shown as lighter pixels (metastatic) compared to darker lower ratio pixels for the non-metastic nodes.

FIG. 7 exemplifies results from an ex vivo mouse tissue activity assay. SDM-23 was incubated with activated tumor and normal thigh muscle tissue homogenates. Enzymatic activity from the tissues resulted in SDM-23 cleavage and generated a large FRET emission ratio increase (labeled primary tumor). The ratio increase was the result of SDM cleavage. Normal muscle tissue showed no cleavage activity of SDM-23.

FIG. 8 exemplifies FRET emission ratio data an ex vivo human tissue assay. SDM-25 was incubated with normal human breast and cancerous human breast tissue (WD2808, WD2821, WD2815, WD2817, WD2824) homogenates. Enzymatic activity and SDM-25 cleavage was found to be significantly greater in cancerous human breast tissue compared to normal human breast tissue (data bar with errors).

FIG. 9 exemplifies FRET emission ratio data from an ex vivo human tissue assay. SDM-25 and SDM-32 were incubated with normal healthy human breast (WD2823) and cancerous human breast tissue (WD2808, WD2815). Enzymatic activity and SDM cleavage was found to be greater in cancerous human breast tissue compared to normal human breast tissue.

FIG. 10 exemplifies a scatter plot of FRET emission ratio data of positive and negative lymph nodes from a mouse metastatic lymph node model that have been treated with SDM-24. Nodes were assigned to be either positive or negative based on analysis of H&E staining by a pathologist.

FIG. 11 exemplifies a ROC curve generated by changing the threshold value used to assign either a positive or negative metastatic prediction from emission ratio data using SDM-24 in metastatic lymph node model. This data show high sensitivity and specificity for diagnosing cancerous and non-cancerous lymph nodes.

DETAILED DESCRIPTION OF THE INVENTION

Selective delivery molecules (SDMs) allow the targeted delivery of therapeutic agents and/or imaging agents to specific cells and/or tissues. In some embodiments, selective delivery molecules comprise (a) a molecular transport or retention sequence (portion B), (b) at least one cargo moiety (portion D) bound to portion A, B, or X, (c) X a linker, and (d) a macromolecular carrier and (e) an acidic sequence (portion A) which is effective to inhibit or prevent the uptake into cells or tissue retention. In some embodiments, cleavage of X linker, which allows the separation of portion A from portion B, is effective to allow the uptake or retention of portion B and the attached cargo into cells and tissue. However, selective delivery molecules may be subject to rapid pharmacokinetic clearance with short plasma half-life, broad distribution, and slow wash out from multiple non-target tissues with non-specific uptake. Thus, there is a need for a selective delivery molecule with increased in vivo circulation, accumulation in target tissue relative to non-target tissue, modulated extravasation selectivity, and modulated bio-distribution. For imaging agents, there is a need for increased contrast in target tissue relative to background tissue.

CERTAIN DEFINITIONS

As used herein, the following terms have the meanings ascribed to them unless specified otherwise.

As used herein, the term “targeting molecule” refers to any agent (e.g., peptide, protein, nucleic acid polymer, aptamer, or small molecule) that associates with (e.g., binds to) a target of interest. The target of interest may be a tissue, a cell, a cellular structure (e.g., an organelle), a protein, a peptide, a polysaccharide, or a nucleic acid polymer. In some embodiments, the targeting molecule is any agent that associates with (e.g., binds to) one or more cancer cells of a subject.

The terms “polypeptide,” “peptide” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to naturally occurring amino acid polymers as well as amino acid polymers in which one or more amino acid residues is a non-naturally occurring amino acid (e.g., an amino acid analog). The terms encompass amino acid chains of any length, including full length proteins (i.e., antigens), wherein the amino acid residues are linked by covalent peptide bonds. As used herein, the terms “peptide” refers to a polymer of amino acid residues typically ranging in length from 2 to about 50 residues. In certain embodiments the peptide ranges in length from about 2, 3, 4, 5, 7, 9, 10, or 11 residues to about 50, 45, 40, 45, 30, 25, 20, or 15 residues. In certain embodiments the peptide ranges in length from about 8, 9, 10, 11, or 12 residues to about 15, 20 or 25 residues. Where an amino acid sequence is provided herein, L-, D-, or beta amino acid versions of the sequence are also contemplated as well as retro, inversion, and retro-inversion isoforms. Peptides also include amino acid polymers in which one or more amino acid residues is an artificial chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers. In addition, the term applies to amino acids joined by a peptide linkage or by other modified linkages (e.g., where the peptide bond is replaced by an α-ester, a β-ester, a thioamide, phosphonamide, carbamate, hydroxylate, and the like (see, e.g., Spatola, (1983) Chem. Biochem. Amino Acids and Proteins 7: 267-357), where the amide is replaced with a saturated amine (see, e.g., Skiles et al., U.S. Pat. No. 4,496,542, which is incorporated herein by reference, and Kaltenbronn et al., (1990) Pp. 969-970 in Proc. 11th American Peptide Symposium, ESCOM Science Publishers, The Netherlands, and the like)).

The term “amino acid” refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, γ-carboxyglutamate, and O-phosphoserine Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an α carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid Amino acids are either D amino acids of L amino acids.

Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes.

One of skill will recognize that individual substitutions, deletions or additions to a peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a “conservatively modified variant” where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles of the invention.

As used herein, the term “label” refers to a molecule that facilitates the visualization and/or detection of a targeting molecule disclosed herein. In some embodiments, the label is a fluorescent moiety.

The phrase “specifically binds” when referring to the interaction between a targeting molecule disclosed herein and a target (e.g., purified protein, cancer cells or cancerous tissue, tumor, or metastatic lesion, metastases, or lymph node or metastatic lymph node), refers to the formation of a high affinity bond between the targeting molecule and the target. Further, the term means that the targeting molecule has low affinity for non-targets.

“Selective binding,” “selectivity,” and the like refers to the preference of an agent to interact with one molecule as compared to another. Preferably, interactions between a targeting molecule disclosed herein and a target are both specific and selective. Note that in some embodiments an agent is designed to “specifically bind” and “selectively bind” two distinct, yet similar targets without binding to other undesirable targets

The terms “individual,” “patient,” or “subject” are used interchangeably. As used herein, they mean any mammal (i.e. species of any orders, families, and genus within the taxonomic classification animalia: chordata: vertebrata: mammalia). In some embodiments, the mammal is a human. None of the terms require or are limited to situation characterized by the supervision (e.g. constant or intermittent) of a health care worker (e.g. a doctor, a registered nurse, a nurse practitioner, a physician's assistant, an orderly, or a hospice worker).

The terms “administer,” “administering”, “administration,” and the like, as used herein, refer to the methods that may be used to enable delivery of agents or compositions to the desired site of biological action. These methods include, but are not limited to parenteral injection (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular, intravascular, intrathecal, intravitreal, infusion, or local). Administration techniques that are optionally employed with the agents and methods described herein, include e.g., as discussed in Goodman and Gilman, The Pharmacological Basis of Therapeutics, current ed.; Pergamon; and Remington's, Pharmaceutical Sciences (current edition), Mack Publishing Co., Easton, Pa.

The term “pharmaceutically acceptable” as used herein, refers to a material that does not abrogate the biological activity or properties of the agents described herein, and is relatively nontoxic (i.e., the toxicity of the material significantly outweighs the benefit of the material). In some instances, a pharmaceutically acceptable material may be administered to an individual without causing significant undesirable biological effects or significantly interacting in a deleterious manner with any of the components of the composition in which it is contained.

The term “surgery” as used herein, refers to any method that may be used to investigate, manipulate, change, or cause an effect in a tissue by a physical intervention. These methods include, but are not limited to open surgery, endoscopic surgery, laparoscopic surgery, minimally invasive surgery, robotic surgery, and any procedures that may affect a cancerous tissue such as tumor resection, cancer tissue ablation, cancer staging, cancer diagnosis, lymph node staging, sentinel lymph node detection, or cancer treatment.

The term “guided surgery” as used herein, refers to any surgical procedure where the surgeon employs an imaging agent to guide the surgery.

The term “cancer” as used herein, refers to any disease involving uncontrolled growth or proliferation cellsin the human body. Cancers may further be characterized by the ability of cells to migrate from the original site and spread to distant sites (i.e., metastasize). Cancers may be sarcomas, carcinomas, lymphomas, leukemias, blastomas, or germ cell tumors. Cancers may occur in a variety of tissues including but not limited to lung, breast, ovaries, colon, esophagus, rectum, bone, prostate, brain, pancreas, bladder, kidney, liver, blood cells, lymph nodes, and stomach.

Selective Delivery Molecules

Disclosed herein, in certain embodiments, are selective delivery molecule of Formula I, having the structure: [D_(A)-c _(A)]-A-[c _(M)-M]-X-B-[c _(B)-D_(B)]  Formula I wherein,

-   -   X is a cleavable linker;     -   A is a peptide with a sequence comprising 5 to 9 acidic amino         acids;     -   B is a peptide with a sequence comprising 7 to 9 basic amino         acids;     -   c_(A), c_(B), and c_(M) each independently comprise 0-1 amino         acid;     -   M is a macromolecule; and     -   D_(A) and D_(B) are each independently selected from an imaging         agent and a therapeutic; and         wherein [c_(M)-M] is bound to at any position on A or X,         [D_(A)-c_(A)] is bound to any amino acid on A, and [c_(B)-D_(B)]         is bound to any amino acid on B.         In some embodiments, A and B do not have an equal number of         acidic and basic amino acids. In some embodiments, the number of         basic amino acids in B is greater than the number of acidic         amino acids in A. In some embodiments, A is a peptide comprising         5 or 9 consecutive glutamates (SEQ ID NO: 3). In some         embodiments, B is a peptide comprising 8 or 9 consecutive         arginines (SEQ ID NO: 4). In some embodiments, A is a peptide         comprising 5 or 9 consecutive glutamates (SEQ ID NO: 3) and B is         a peptide comprising 8 or 9 consecutive arginines (SEQ ID NO:         4). In some embodiments, A is a peptide comprising 5 consecutive         glutamates (SEQ ID NO: 5) and B is a peptide comprising 8         consecutive arginines (SEQ ID NO: 6). In some embodiments,         c_(A), c_(B), and c_(M) are each independently a 0-1 amino acid.         In some embodiments, c_(A), c_(B), and c_(M) are each         independently selected from a naturally-occurring amino acid or         a non-naturally-occurring amino acid. In some embodiments,         c_(A), c_(B), and c_(M) are each independently selected from a D         amino acid, a L amino acid, an α-amino acid, a β-amino acid, or         a γ-amino acid. In some embodiments, c_(A), c_(B), and c_(M) are         each independently selected from any amino acid having a free         thiol group, any amino acid having a N-terminal amine group, and         any amino acid with a side chain capable of forming an oxime or         hydrazone bond upon reaction with a hydroxylamine or hydrazine         group. In some embodiments, c_(A), c_(B), and c_(M) are each         independently selected from D-cysteine, D-glutamate, lysine, and         para-4-acetyl L-phenylalanine. In some embodiments, c_(B) is any         amino acid having a free thiol group. In some embodiments, c_(B)         is D-cysteine. In some embodiments, c_(A) is any amino acid         having a N-terminal amine group. In some embodiments, c_(A) is         D-glutamate. In some embodiments, c_(A) is lysine. In some         embodiments, c_(M) is any amino acid with a side chain capable         of forming an oxime or hydrazone bond upon reaction with a         hydroxylamine or hydrazine group. In some embodiments, c_(M) is         para-4-acetyl L-phenylalanine. In some embodiments, X is         cleavable by a protease. In some embodiments, X is cleavable by         a matrix metalloproteinase. In some embodiments, X comprises an         amino acid sequence that is cleavable by MMP2, MMP7, MMP9, or         MMP14. In some embodiments, X comprises a peptide linkage. In         some embodiments, X comprises an amino acid sequence selected         from: PLGLAG (SEQ ID NO: 2), PLG-C(me)-AG (SEQ ID NO: 1),         RPLALWRS (SEQ ID NO: 7), ESPAYYTA (SEQ ID NO: 8), DPRSFL (SEQ ID         NO: 9), PPRSFL (SEQ ID NO: 10), RLQLKL (SEQ ID NO: 11), and         RLQLK(Ac) (SEQ ID NO: 12). In some embodiments, X comprises the         amino acid sequence PLGLAG (SEQ ID NO: 2). In some embodiments,         X comprises the amino acid sequence PLG-C(me)-AG (SEQ ID NO: 1).         In some embodiments, X comprises the amino acid sequence         RPLALWRS (SEQ ID NO: 7). In some embodiments, X comprises the         amino acid sequence DPRSFL (SEQ ID NO: 9). In some embodiments,         X comprises the amino acid sequence RLQLKL (SEQ ID NO: 11). In         some embodiments, X comprises the amino acid sequence RLQLK(Ac)         (SEQ ID NO: 12). In some embodiments, M is selected from a         protein, a natural polymer, a synthetic polymer, or a dendrimer.         In some embodiments, M is selected from dextran, a PEG polymer,         albumin, or a combination thereof. In some embodiments, M is a         PEG. In some embodiments, M is selected from PEG 5 kDa, PEG 12         kDa, PEG 20 kDa, PEG 30 kDa, and PEG40 kDa. In some embodiments,         D_(A) and D_(B) are a pair of acceptor and donor fluorescent         moieties that are capable of undergoing Försters/fluorescence         resonance energy transfer with the other. In some embodiments,         D_(A) and D_(B) are Cy5 and Cy7. In some embodiments, D_(A) and         D_(B) are Cy5 and IRDye750. In some embodiments, D_(A) and D_(B)         are Cy5 and IRDye800. In some embodiments, D_(A) and D_(B) are         Cy5 and ICG. In some embodiments, D_(A) and D_(B) are a         fluorescent moiety and a fluorescence-quenching moiety. In some         embodiments, the molecule of Formula I is: SDM-14, SDM-15,         SDM-23, SDM-24, SDM-25, SDM-26, SDM-27, SDM-32, or SDM-35.

Disclosed herein, in certain embodiments, are selective delivery molecules of Formula I, having the structure: [D_(A)-c _(A)]-A-[c _(M)-M]-X-B-[c _(B)-D_(B)]  Formula I wherein,

-   -   X is a cleavable linker;     -   A is a peptide with a sequence comprising 5 to 9 acidic amino         acids;     -   B is a peptide with a sequence comprising 7 to 9 basic amino         acids;     -   c_(A), c_(B), and c_(M) each independently comprise 0-1 amino         acid;     -   M is a polyethylene glycol (PEG) polymer; and     -   D_(A) and D_(B) are each independently an imaging agent; and     -   wherein [c_(M)-M] is bound to at any position on A or X,         [D_(A)-c_(A)] is bound to any amino acid on A, and [c_(B)-D_(B)]         is bound to any amino acid on B.         In some embodiments, A and B do not have an equal number of         acidic and basic amino acids. In some embodiments, the number of         basic amino acids in B is greater than the number of acidic         amino acids in A. In some embodiments, A is a peptide comprising         5 or 9 consecutive glutamates (SEQ ID NO: 3). In some         embodiments, B is a peptide comprising 8 or 9 consecutive         arginines (SEQ ID NO: 4). In some embodiments, A is a peptide         comprising 5 or 9 consecutive glutamates (SEQ ID NO: 3) and B is         a peptide comprising 8 or 9 consecutive arginines (SEQ ID NO:         4). In some embodiments, A is a peptide comprising 5 consecutive         glutamates (SEQ ID NO: 5) and B is a peptide comprising 8         consecutive arginines (SEQ ID NO: 6). In some embodiments,         c_(A), c_(B), and c_(M) are each independently a 0-1 amino acid.         In some embodiments, c_(A), c_(B), and c_(M) are each         independently selected from a naturally-occurring amino acid or         a non-naturally-occurring amino acid. In some embodiments,         c_(A), c_(B), and c_(M) are each independently selected from a D         amino acid, a L amino acid, an α-amino acid, a β-amino acid, or         a γ-amino acid. In some embodiments, c_(A), c_(B), and c_(M) are         each independently selected from any amino acid having a free         thiol group, any amino acid having a N-terminal amine group, and         any amino acid with a side chain capable of forming an oxime or         hydrazone bond upon reaction with a hydroxylamine or hydrazine         group. In some embodiments, c_(A), c_(B), and c_(M) are each         independently selected from D-cysteine, D-glutamate, lysine, and         para-4-acetyl L-phenylalanine. In some embodiments, c_(B) is any         amino acid having a free thiol group. In some embodiments, c_(B)         is D-cysteine. In some embodiments, c_(A) is any amino acid         having a N-terminal amine group. In some embodiments, c_(A) is         D-glutamate. In some embodiments, c_(A) is lysine. In some         embodiments, c_(M) is any amino acid with a side chain capable         of forming an oxime or hydrazone bond upon reaction with a         hydroxylamine or hydrazine group. In some embodiments, c_(M) is         para-4-acetyl L-phenylalanine. In some embodiments, X is         cleavable by a protease. In some embodiments, X is cleavable by         a matrix metalloproteinase. In some embodiments, X comprises an         amino acid sequence that is cleavable by MMP2, MMP7, MMP9, or         MMP14. In some embodiments, X comprises a peptide linkage. In         some embodiments, X comprises an amino acid sequence selected         from: PLGLAG (SEQ ID NO: 2), PLG-C(me)-AG (SEQ ID NO: 1),         RPLALWRS (SEQ ID NO: 7), ESPAYYTA (SEQ ID NO: 8), DPRSFL (SEQ ID         NO: 9), PPRSFL (SEQ ID NO: 10), RLQLKL (SEQ ID NO: 11), and         RLQLK(Ac) (SEQ ID NO: 12). In some embodiments, X comprises the         amino acid sequence PLGLAG (SEQ ID NO: 2). In some embodiments,         X comprises the amino acid sequence PLG-C(me)-AG (SEQ ID NO: 1).         In some embodiments, X comprises the amino acid sequence         RPLALWRS (SEQ ID NO: 7). In some embodiments, X comprises the         amino acid sequence DPRSFL (SEQ ID NO: 9). In some embodiments,         X comprises the amino acid sequence PPRSFL (SEQ ID NO: 10). In         some embodiments, X comprises the amino acid sequence RLQLKL         (SEQ ID NO: 11). In some embodiments, X comprises the amino acid         sequence RLQLK(Ac) (SEQ ID NO: 12). In some embodiments, D_(A)         and D_(B) are a pair of acceptor and donor fluorescent moieties         that are capable of undergoing Försters/fluorescence resonance         energy transfer with the other. In some embodiments, D_(A) and         D_(B) are Cy5 and Cy7. In some embodiments, D_(A) and D_(B) are         Cy5 and IRDye750. In some embodiments, D_(A) and D_(B) are Cy5         and IRDye800. In some embodiments, D_(A) and D_(B) are Cy5 and         ICG. In some embodiments, D_(A) and D_(B) are a fluorescent         moiety and a fluorescence-quenching moiety. In some embodiments,         the molecule of Formula I is: SDM-14, SDM-15, SDM-23, SDM-24,         SDM-25, SDM-26, SDM-27, SDM-32; or SDM-35.

Disclosed herein, in certain embodiments, are selective delivery molecules of Formula I, having the structure: [D_(A)-c _(A)]-A-[c _(M)-M]-X-B-[c _(B)-D_(B)]  Formula I wherein,

-   -   X is a peptide linker cleavable by a matrix metalloproteinase;     -   A is a peptide with a sequence comprising 5 or 9 consecutive         glutamates (SEQ ID NO: 3);     -   B is a peptide with a sequence comprising 8 or 9 consecutive         arginines (SEQ ID NO: 4);     -   c_(A), c_(B), and c_(M) each independently comprise 0-1 amino         acid;     -   M is a polyethylene glycol (PEG) polymer; and     -   D_(A) and D_(B) are a pair of acceptor and donor fluorescent         moieties that are capable of undergoing Försters/fluorescence         resonance energy transfer with the other; and         wherein [c_(M)-M] is bound to at any position on A or X,         [D_(A)-c_(A)] is bound to any amino acid on A, and [c_(B)-D_(B)]         is bound to any amino acid on B.         In some embodiments, A and B do not have an equal number of         acidic and basic amino acids. In some embodiments, c_(A), c_(B),         and c_(M) are each independently selected from any amino acid         having a free thiol group, any amino acid having a N-terminal         amine group, and any amino acid with a side chain capable of         forming an oxime or hydrazone bond upon reaction with a         hydroxylamine or hydrazine group. In some embodiments, c_(A),         c_(B), and c_(M) are each independently a 0-1 amino acid. In         some embodiments, c_(A), c_(B), and c_(M) are each independently         selected from D-cysteine, D-glutamate, lysine, and para-4-acetyl         L-phenylalanine. In some embodiments, c_(B) is any amino acid         having a free thiol group. In some embodiments, c_(B) is         D-cysteine. In some embodiments, c_(A) is any amino acid having         a N-terminal amine group. In some embodiments, c_(A) is         D-glutamate. In some embodiments, c_(M) is any amino acid with a         side chain capable of forming an oxime or hydrazone bond upon         reaction with a hydroxylamine or hydrazine group. In some         embodiments, c_(M) is para-4-acetyl L-phenylalanine. In some         embodiments, X comprises the amino acid sequence PLGLAG (SEQ ID         NO: 2). In some embodiments, X comprises the amino acid sequence         PLG-C(me)-AG (SEQ ID NO: 1). In some embodiments, D_(A) and         D_(B) are Cy5 and Cy7. In some embodiments, D_(A) and D_(B) are         Cy5 and Cy7.

Disclosed herein, in certain embodiments, are selective delivery molecules of Formula I, having the structure: [D_(A)-c _(A)]-A-[c _(M)-M]-X-B-[c _(B)-D_(B)]  Formula I wherein,

-   -   X is a peptide linker cleavable by a matrix metalloproteinase;     -   A is a peptide with a sequence comprising 5 consecutive         glutamates (SEQ ID NO: 5);     -   B is a peptide with a sequence comprising 8 consecutive         arginines (SEQ ID NO: 6);     -   c_(A), c_(B), and c_(M) each independently comprise 0-1 amino         acid;     -   M is a polyethylene glycol (PEG) polymer; and     -   D_(A) and D_(B) are a pair of acceptor and donor fluorescent         moieties that are capable of undergoing Försters/fluorescence         resonance energy transfer with the other; and         wherein [c_(M)-M] is bound to at any position on A or X,         [D_(A)-c_(A)] is bound to any amino acid on A, and [c_(B)-D_(B)]         is bound to any amino acid on B.         In some embodiments, c_(A), c_(B), and c_(M) are each         independently a 0-1 amino acid. In some embodiments, c_(A),         c_(B), and c_(M) are each independently selected from any amino         acid having a free thiol group, any amino acid having a         N-terminal amine group, and any amino acid with a side chain         capable of forming an oxime or hydrazone bond upon reaction with         a hydroxylamine or hydrazine group. In some embodiments, c_(A),         c_(B), and c_(M) are each independently selected from         D-cysteine, D-glutamate, lysine, and para-4-acetyl         L-phenylalanine. In some embodiments, c_(B) is any amino acid         having a free thiol group. In some embodiments, c_(B) is         D-cysteine. In some embodiments, c_(A) is any amino acid having         a N-terminal amine group. In some embodiments, c_(A) is         D-glutamate. In some embodiments, c_(M) is any amino acid with a         side chain capable of forming an oxime or hydrazone bond upon         reaction with a hydroxylamine or hydrazine group. In some         embodiments, c_(M) is para-4-acetyl L-phenylalanine. In some         embodiments, X comprises the amino acid sequence PLGLAG (SEQ ID         NO: 2). In some embodiments, X comprises the amino acid sequence         PLG-C(me)-AG (SEQ ID NO: 1). In some embodiments, X comprises         the amino acid sequence RPLALWRS (SEQ ID NO: 7). In some         embodiments, D_(A) and D_(B) are Cy5 and Cy7. In some         embodiments, D_(A) and D_(B) are Cy5 and IRDye750. In some         embodiments, D_(A) and D_(B) are Cy5 and IRDye800. In some         embodiments, D_(A) and D_(B) are Cy5 and ICG.

Disclosed herein, in certain embodiments, are selective delivery molecules of Formula I, having the structure: [D_(A)-c _(A)]-A-[c _(M)-M]-X-B-[c _(B)-D_(B)]  Formula I wherein,

-   -   X is a peptide linker cleavable by a matrix metalloproteinase;     -   A is a peptide with a sequence comprising 9 consecutive         glutamates (SEQ ID NO: 13);     -   B is a peptide with a sequence comprising 9 consecutive         arginines (SEQ ID NO: 14);     -   c_(A), c_(B), and c_(M) each independently comprise 0-1 amino         acid;     -   M is a polyethylene glycol (PEG) polymer; and     -   D_(A) and D_(B) are a pair of acceptor and donor fluorescent         moieties that are capable of undergoing Försters/fluorescence         resonance energy transfer with the other; and         wherein [c_(M)-M] is bound to at any position on A or X,         [D_(A)-c_(A)] is bound to any amino acid on A, and [c_(B)-D_(B)]         is bound to any amino acid on B.         In some embodiments, c_(A), c_(B), and c_(M) are each         independently a 0-1 amino acid. In some embodiments, c_(A),         c_(B), and c_(M) are each independently selected from any amino         acid having a free thiol group, any amino acid having a         N-terminal amine group, and any amino acid with a side chain         capable of forming an oxime or hydrazone bond upon reaction with         a hydroxylamine or hydrazine group. In some embodiments, c_(A),         c_(B), and c_(M) are each independently selected from         D-cysteine, D-glutamate, lysine, and para-4-acetyl         L-phenylalanine. In some embodiments, c_(B) is any amino acid         having a free thiol group. In some embodiments, c_(B) is         D-cysteine. In some embodiments, c_(A) is any amino acid having         a N-terminal amine group. In some embodiments, c_(A) is         D-glutamate. In some embodiments, c_(M) is any amino acid with a         side chain capable of forming an oxime or hydrazone bond upon         reaction with a hydroxylamine or hydrazine group. In some         embodiments, c_(M) is para-4-acetyl L-phenylalanine. In some         embodiments, X comprises the amino acid sequence PLGLAG (SEQ ID         NO: 2). In some embodiments, X comprises the amino acid sequence         PLG-C(me)-AG (SEQ ID NO: 1). In some embodiments, X comprises         the amino acid sequence RPLALWRS (SEQ ID NO: 7). In some         embodiments, D_(A) and D_(B) are Cy5 and Cy7. In some         embodiments, D_(A) and D_(B) are Cy5 and IRDye750. In some         embodiments, D_(A) and D_(B) are Cy5 and IRDye800. In some         embodiments, D_(A) and D_(B) are Cy5 and ICG.

Disclosed herein, in certain embodiments, are selective delivery molecules according to SDM-14.

Disclosed herein, in certain embodiments, are selective delivery molecules according to SDM-15.

Disclosed herein, in certain embodiments, are selective delivery molecules according to SDM-23.

Disclosed herein, in certain embodiments, are selective delivery molecules according to SDM-24.

Disclosed herein, in certain embodiments, are selective delivery molecules according to SDM-25.

Disclosed herein, in certain embodiments, are selective delivery molecules according to SDM-26.

Disclosed herein, in certain embodiments, are selective delivery molecules according to SDM-27.

Disclosed herein, in certain embodiments, are selective delivery molecules according to SDM-32.

Disclosed herein, in certain embodiments, are selective delivery molecules according to SDM-35.

Disclosed herein, in certain embodiments, are peptides according to Peptide P-3.

Disclosed herein, in certain embodiments, are selective delivery molecules according to SDM-14.

Disclosed herein, in certain embodiments, are selective delivery molecules according to SDM-15.

Disclosed herein, in certain embodiments, are selective delivery molecules according to SDM-23.

Disclosed herein, in certain embodiments, are selective delivery molecules according to SDM-24.

Disclosed herein, in certain embodiments, are selective delivery molecules according to SDM-25.

Disclosed herein, in certain embodiments, are selective delivery molecules according to SDM-26.

Disclosed herein, in certain embodiments, are selective delivery molecules according to SDM-27.

Disclosed herein, in certain embodiments, are selective delivery molecules according to SDM-32.

Disclosed herein, in certain embodiments, are selective delivery molecules according to SDM-35.

Portion A

In some embodiments, A is a peptide with a sequence comprising 2 to 20 acidic amino acids. In some embodiments, peptide portion A comprises between about 2 to about 20 acidic amino acids. In some embodiments, peptide portion A comprises between about 5 to about 20 acidic amino acids. In some embodiments, A has a sequence comprising 5 to 9 acidic amino acids. In some embodiments, A has a sequence comprising 5 to 8 acidic amino acids. In some embodiments, A has a sequence comprising 5 to 7 acidic amino acids. In some embodiments, A has a sequence comprising 5 acidic amino acids. In some embodiments, A has a sequence comprising 6 acidic amino acids. In some embodiments, A has a sequence comprising 7 acidic amino acids. In some embodiments, A has a sequence comprising 8 acidic amino acids. In some embodiments, A has a sequence comprising 9 acidic amino acids.

In some embodiments, peptide portion A comprises between about 2 to about 20 consecutive acidic amino acids. In some embodiments, peptide portion A comprises between about 5 to about 20 consecutive acidic amino acids. In some embodiments, A has a sequence comprising 5 to 9 consecutive acidic amino acids. In some embodiments, A has a sequence comprising 5 to 8 consecutive acidic amino acids. In some embodiments, A has a sequence comprising 5 to 7 consecutive acidic amino acids. In some embodiments, A has a sequence comprising 5 consecutive acidic amino acids. In some embodiments, A has a sequence comprising 6 consecutive acidic amino acids. In some embodiments, A has a sequence comprising 7 consecutive acidic amino acids. In some embodiments, A has a sequence comprising 8 consecutive acidic amino acids. In some embodiments, A has a sequence comprising 9 consecutive acidic amino acids.

In some embodiments, peptide portion A comprises between about 2 to about 20 acidic amino acids selected from, aspartates and glutamates. In some embodiments, peptide portion A comprises between about 5 to about 20 acidic amino acids selected from, aspartates and glutamates. In some embodiments, A has a sequence comprising 5 to 9 acidic amino acids selected from, aspartates and glutamates. In some embodiments, A has a sequence comprising 5 to 8 acidic amino acids selected from, aspartates and glutamates. In some embodiments, A has a sequence comprising 5 to 7 acidic amino acids selected from, aspartates and glutamates. In some embodiments, A has a sequence comprising 5 acidic amino acids selected from, aspartates and glutamates. In some embodiments, A has a sequence comprising 6 acidic amino acids selected from, aspartates and glutamates. In some embodiments, A has a sequence comprising 7 acidic amino acids selected from, aspartates and glutamates. In some embodiments, A has a sequence comprising 8 acidic amino acids selected from, aspartates and glutamates. In some embodiments, A has a sequence comprising 9 acidic amino acids selected from, aspartates and glutamates.

In some embodiments, peptide portion A comprises between about 2 to about 20 consecutive acidic amino acids selected from, aspartates and glutamates. In some embodiments, peptide portion A comprises between about 5 to about 20 consecutive acidic amino acids selected from, aspartates and glutamates. In some embodiments, A has a sequence comprising 5 to 9 consecutive acidic amino acids selected from, aspartates and glutamates. In some embodiments, A has a sequence comprising 5 to 8 consecutive acidic amino acids selected from, aspartates and glutamates. In some embodiments, A has a sequence comprising 5 to 7 consecutive acidic amino acids selected from, aspartates and glutamates. In some embodiments, A has a sequence comprising 5 consecutive acidic amino acids selected from, aspartates and glutamates. In some embodiments, A has a sequence comprising 6 consecutive acidic amino acids selected from, aspartates and glutamates. In some embodiments, A has a sequence comprising 7 consecutive acidic amino acids selected from, aspartates and glutamates. In some embodiments, A has a sequence comprising 8 consecutive acidic amino acids selected from, aspartates and glutamates. In some embodiments, A has a sequence comprising 9 consecutive acidic amino acids selected from, aspartates and glutamates.

In some embodiments, peptide portion A comprises between about 2 to about 20 glutamates. In some embodiments, peptide portion A comprises between about 5 to about 20 glutamates. In some embodiments, A has a sequence comprising 5 to 9 glutamates. In some embodiments, A has a sequence comprising 5 to 8 glutamates. In some embodiments, A has a sequence comprising 5 to 7 glutamates. In some embodiments, A has a sequence comprising 5 glutamates. In some embodiments, A has a sequence comprising 6 glutamates. In some embodiments, A has a sequence comprising 7 glutamates. In some embodiments, A has a sequence comprising 8 glutamates. In some embodiments, A has a sequence comprising 9 glutamates.

In some embodiments, peptide portion A comprises between about 2 to about 20 consecutive glutamates (SEQ ID NO: 15). In some embodiments, peptide portion A comprises between about 5 to about 20 consecutive glutamates (SEQ ID NO: 16). In some embodiments, A has a sequence comprising 5 to 9 consecutive glutamates (SEQ ID NO: 41). In some embodiments, A has a sequence comprising 5 to 8 consecutive glutamates (SEQ ID NO: 17). In some embodiments, A has a sequence comprising 5 to 7 consecutive glutamates (SEQ ID NO: 18). In some embodiments, A has a sequence comprising 5 consecutive glutamates (SEQ ID NO: 5). In some embodiments, A has a sequence comprising 6 consecutive glutamates (SEQ ID NO: 19). In some embodiments, A has a sequence comprising 7 consecutive glutamates (SEQ ID NO: 20). In some embodiments, A has a sequence comprising 8 consecutive glutamates (SEQ ID NO: 21). In some embodiments, A has a sequence comprising 9 consecutive glutamates (SEQ ID NO: 13).

In some embodiments, portion A comprises 5 consecutive glutamates (SEQ ID NO: 5) (i.e., EEEEE (SEQ ID NO: 5) or eeeee). In some embodiments, portion A comprises 9 consecutive glutamates (SEQ ID NO: 13) (i.e., EEEEEEEEE (SEQ ID NO: 13) or eeeeeeeee).

An acidic portion A may include amino acids that are not acidic. Acidic portion A may comprise other moieties, such as negatively charged moieties. In embodiments of a selective delivery molecule disclosed herein, an acidic portion A may be a negatively charged portion, preferably having about 2 to about 20 negative charges at physiological pH that does not include an amino acid.

In some embodiments, the amount of negative charge in portion A is approximately the same as the amount of positive charge in portion B. In some embodiments, the amount of negative charge in portion A is not the same as the amount of positive charge in portion B. In some embodiments, improved tissue uptake is seen in a selective delivery molecule wherein the amount of negative charge in portion A is not the same as the amount of positive charge in portion B. In some embodiments, improved solubility is observed in a selective delivery molecule wherein the amount of negative charge in portion A is not the same as the amount of positive charge in portion B. In some embodiments, faster tissue uptake is seen in a selective delivery molecule wherein the amount of negative charge in portion A is not the same as the amount of positive charge in portion B. In some embodiments, greater tissue uptake is seen in a selective delivery molecule wherein the amount of negative charge in portion A is not the same as the amount of positive charge in portion B.

Portion A is either L-amino acids or D-amino acids. In embodiments of the invention, D-amino acids are preferred in order to minimize immunogenicity and nonspecific cleavage by background peptidases or proteases. Cellular uptake of oligo-D-arginine sequences is known to be as good as or better than that of oligo-L-arginines

It will be understood that portion A may include non-standard amino acids, such as, for example, hydroxylysine, desmosine, isodesmosine, or other non-standard amino acids. Portion A may include modified amino acids, including post-translationally modified amino acids such as, for example, methylated amino acids (e.g., methyl histidine, methylated forms of lysine, etc.), acetylated Amino acids, amidated amino acids, formylated amino acids, hydroxylated amino acids, phosphorylated amino acids, or other modified amino acids. Portion A may also include peptide mimetic moieties, including portions linked by non-peptide bonds and amino acids linked by or to non-amino acid portions.

The Selective Delivery Molecules disclosed herein are effective where A is at the amino terminus or where A is at the carboxy terminus, i.e., either orientation of the peptide bonds is permissible.

Portion B

In some embodiments, B is a peptide with a sequence comprising 5 to 15 basic amino acids. In some embodiments, peptide portion B comprises between about 5 to about 20 basic amino acids. In some embodiments, peptide portion B comprises between about 5 to about 12 basic amino acids. In some embodiments, peptide portion B comprises between about 7 to about 9 basic amino acids. In some embodiments, peptide portion B comprises between about 7 to about 8 basic amino acids. In some embodiments, peptide portion B comprises 9 basic amino acids. In some embodiments, peptide portion B comprises 8 basic amino acids. In some embodiments, peptide portion B comprises 7 basic amino acids.

In some embodiments, peptide portion B comprises between about 5 to about 20 consecutive basic amino acids. In some embodiments, peptide portion B comprises between about 5 to about 12 consecutive basic amino acids. In some embodiments, peptide portion B comprises between about 7 to about 9 consecutive basic amino acids. In some embodiments, peptide portion B comprises between about 7 to about 8 consecutive basic amino acids. In some embodiments, peptide portion B comprises 9 consecutive basic amino acids. In some embodiments, peptide portion B comprises 8 consecutive basic amino acids. In some embodiments, peptide portion B comprises 7 consecutive basic amino acids.

In some embodiments, peptide portion B comprises between about 5 to about 20 basic amino acids selected from arginines, histidines, and lysines. In some embodiments, peptide portion B comprises between about 5 to about 12 basic amino acids selected from arginines, histidines, and lysines. In some embodiments, peptide portion B comprises between about 7 to about 9 basic amino acids selected from arginines, histidines, and lysines. In some embodiments, peptide portion B comprises between about 7 to about 8 basic amino acids selected from arginines, histidines, and lysines. In some embodiments, peptide portion B comprises 9 basic amino acids selected from arginines, histidines, and lysines. In some embodiments, peptide portion B comprises 8 basic amino acids selected from arginines, histidines, and lysines. In some embodiments, peptide portion B comprises 7 basic amino acids selected from arginines, histidines, and lysines.

In some embodiments, peptide portion B comprises between about 5 to about 20 consecutive basic amino acids selected from arginines, histidines, and lysines. In some embodiments, peptide portion B comprises between about 5 to about 12 consecutive basic amino acids selected from arginines, histidines, and lysines. In some embodiments, peptide portion B comprises between about 7 to about 9 consecutive basic amino acids selected from arginines, histidines, and lysines. In some embodiments, peptide portion B comprises between about 7 to about 8 consecutive basic amino acids selected from arginines, histidines, and lysines. In some embodiments, peptide portion B comprises 9 consecutive basic amino acids selected from arginines, histidines, and lysines. In some embodiments, peptide portion B comprises 8 consecutive basic amino acids selected from arginines, histidines, and lysines. In some

In some embodiments, peptide portion B comprises between about 5 to about 20 arginines. In some embodiments, peptide portion B comprises between about 5 to about 12 arginines. In some embodiments, peptide portion B comprises between about 7 to about 9 arginines. In some embodiments, peptide portion B comprises between about 7 to about 8 arginines. In some embodiments, peptide portion B comprises 9 arginines. In some embodiments, peptide portion B comprises 8 arginines. In some embodiments, peptide portion B comprises 7 arginines.

In some embodiments, peptide portion B comprises between about 5 to about 20 consecutive arginines (SEQ ID NO: 22). In some embodiments, peptide portion B comprises between about 5 to about 12 consecutive arginines (SEQ ID NO: 23). In some embodiments, peptide portion B comprises between about 7 to about 9 consecutive arginines (SEQ ID NO: 24). In some embodiments, peptide portion B comprises between about 7 to about 8 consecutive arginines (SEQ ID NO: 25). In some embodiments, peptide portion B comprises 9 consecutive arginines (SEQ ID NO: 14). In some embodiments, peptide portion B comprises 8 consecutive arginines (SEQ ID NO: 6). In some embodiments, peptide portion B comprises 7 consecutive arginines (SEQ ID NO: 26).

A basic portion B may include amino acids that are not basic. Basic portion B may comprise other moieties, such as positively charged moieties. In embodiments, a basic portion B may be a positively charged portion, preferably having between about 5 and about 20 positive charges at physiological pH, that does not include an amino acid. In some embodiments, the amount of negative charge in portion A is approximately the same as the amount of positive charge in portion B. In some embodiments, the amount of negative charge in portion A is not the same as the amount of positive charge in portion B.

Portion B is either L-amino acids or D-amino acids. In embodiments of the invention, D-amino acids are preferred in order to minimize immunogenicity and nonspecific cleavage by background peptidases or proteases. Cellular uptake of oligo-D-arginine sequences is known to be as good as or better than that of oligo-L-arginines

It will be understood that portion B may include non-standard amino acids, such as, for example, hydroxylysine, desmosine, isodesmosine, or other non-standard amino acids. Portion B may include modified amino acids, including post-translationally modified amino acids such as, for example, methylated amino acids (e.g., methyl histidine, methylated forms of lysine, etc.), acetylated amino acids, amidated amino acids, formylated amino acids, hydroxylated amino acids, phosphorylated amino acids, or other modified amino acids. Portion B may also include peptide mimetic moieties, including portions linked by non-peptide bonds and amino acids linked by or to non-amino acid portions.

In embodiments where X is a peptide cleavable by a protease, it may be preferable to join the C-terminus of X to the N-terminus of B, so that the new amino terminus created by cleavage of X contributes an additional positive charge that adds to the positive charges already present in B.

Conjugation Group (c)

In some embodiments, the cargo (e.g., D_(A) and D_(B)) and the macromolecule carriers (M) are attached indirectly to A-X-B.

In some embodiments, the cargo (e.g., D_(A) and D_(B)) and the macromolecule carriers (M) are attached indirectly to A-X-B by a conjugation group (c_(A), c_(B), and c_(M)). In some embodiments, the cargo (e.g., D_(A) and D_(B)) and the macromolecule carriers (M) are attached indirectly to A-X-B by a reactive conjugation group (c_(A), c_(B), and c_(M)). In some embodiments, the cargo (e.g., D_(A) and D_(B)) and the macromolecule carriers (M) are attached indirectly to A-X-B by an orthogonally reactive conjugation group (c_(A), c_(B), and c_(M)). In some embodiments, c_(A), c_(B), and c_(M) each independently comprise an amino acid. In some embodiments, c_(A), c_(B), and c_(M) each independently comprise 0-10 amino acids. In some embodiments, c_(A), c_(B), and c_(M) each independently comprise 1 amino acid. In some embodiments, c_(A), c_(B), and c_(M) each independently comprise 2 amino acids. In some embodiments, c_(A), c_(B), and c_(M) each independently comprise 3 amino acids. In some embodiments, c_(A), c_(B), and c_(M) each independently comprise 4 amino acids. In some embodiments, c_(A), c_(B), and c_(M) each independently comprise 5 amino acids. In some embodiments, c_(A), c_(B), and c_(M) each independently comprise 6 amino acids. In some embodiments, c_(A), c_(B), and c_(M) each independently comprise 7 amino acids. In some embodiments, c_(A), c_(B), and c_(M) each independently comprise 8 amino acids. In some embodiments, c_(A), c_(B), and c_(M) each independently comprise 9 amino acids. In some embodiments, c_(A), c_(B), and c_(M) each independently comprise 10 amino acids.

In some embodiments, c_(A), c_(B), and c_(M) each independently comprise a derivatized amino acid. In some embodiments, multiple cargos (D) are attached to a derivatized amino acid conjugation group.

In some embodiments, the conjugation group comprises a receptor ligand.

In some embodiments, c_(A), c_(B), and c_(M) each independently comprise a naturally-occurring amino acid or a non-naturally-occurring amino acid. In some embodiments, c_(A), c_(B), and c_(M) each independently comprise from a D amino acid, a L amino acid, an α-amino acid, a B-amino acid, or a γ-amino acid. In some embodiments, c_(A), c_(B), and c_(M) each independently comprise any amino acid having a free thiol group, any amino acid containing a free amine group, any amino acid having a N-terminal amine group, and any amino acid with a side chain capable of forming an oxime or hydrazone bond upon reaction with a hydroxylamine or hydrazine group. In some embodiments, c_(A), c_(B), and c_(M) each independently comprise D-cysteine, D-glutamate, lysine, and para-4-acetyl L-phenylalanine. In some embodiments, c_(B) comprises any amino acid having a free thiol group. In some embodiments, c_(B) comprises D-cysteine. In some embodiments, c_(A) comprises any amino acid having a N-terminal amine group. In some embodiments, c_(A) comprises D-glutamate. In some embodiments, c_(A) comprises lysine. In some embodiments, c_(M) comprises any amino acid with a side chain capable of forming an oxime or hydrazone bond upon reaction with a hydroxylamine or hydrazine group. In some embodiments, c_(M) comprises para-4-acetyl L-phenylalanine.

In some embodiments, c_(A), c_(B), and c_(M) are each independently selected from a naturally-occurring amino acid or a non-naturally-occurring amino acid. In some embodiments, c_(A), c_(B), and c_(M) are each independently selected from a D amino acid, a L amino acid, an α-amino acid, a β-amino acid, or a γ-amino acid. In some embodiments, c_(A), c_(B), and c_(M) are each independently any amino acid having a free thiol group, any amino acid containing a free amine group, any amino acid having a N-terminal amine group, and any amino acid with a side chain capable of forming an oxime or hydrazone bond upon reaction with a hydroxylamine or hydrazine group. In some embodiments, c_(A), c_(B), and c_(M) are each independently selected from: D-cysteine, D-glutamate, lysine, and para-4-acetyl L-phenylalanine. In some embodiments, c_(B) is any amino acid having a free thiol group. In some embodiments, c_(B) is D-cysteine. In some embodiments, c_(A) is any amino acid having a N-terminal amine group. In some embodiments, c_(A) is D-glutamate. In some embodiments, c_(A) is lysine. In some embodiments, c_(M) is any amino acid with a side chain capable of forming an oxime or hydrazone bond upon reaction with a hydroxylamine or hydrazine group. In some embodiments, c_(M) is para-4-acetyl L-phenylalanine.

Cargo (D)

Imaging Agents

In some embodiments, an imaging agent is a dye. In some embodiments, an imaging agent is a fluorescent moiety. In some embodiments, a fluorescent moiety is selected from: a fluorescent protein, a fluorescent peptide, a fluorescent dye, a fluorescent material or a combination thereof.

All fluorescent moieties are encompassed within the term “fluorescent moiety.” Specific examples of fluorescent moieties given herein are illustrative and are not meant to limit the fluorescent moieties for use with the targeting molecules disclosed herein.

Examples of fluorescent dyes include, but are not limited to, xanthenes (e.g., rhodamines, rhodols and fluoresceins, and their derivatives); bimanes; coumarins and their derivatives (e.g., umbelliferone and aminomethyl coumarins); aromatic amines (e.g., dansyl; squarate dyes); benzofurans; fluorescent cyanines; indocarbocyanines; carbazoles; dicyanomethylene pyranes; polymethine; oxabenzanthrane; xanthene; pyrylium; carbostyl; perylene; acridone; quinacridone; rubrene; anthracene; coronene; phenanthrecene; pyrene; butadiene; stilbene; porphyrin; pthalocyanine; lanthanide metal chelate complexes; rare-earth metal chelate complexes; and derivatives of such dyes.

Examples of fluorescein dyes include, but are not limited to, 5-carboxyfluorescein, fluorescein-5-isothiocyanate, fluorescein-6-isothiocyanate and 6-carboxyfluorescein.

Examples of rhodamine dyes include, but are not limited to, tetramethylrhodamine-6-isothiocyanate, 5-carboxytetramethylrhodamine, 5-carboxy rhodol derivatives, tetramethyl and tetraethyl rhodamine, diphenyldimethyl and diphenyldiethyl rhodamine, dinaphthyl rhodamine, rhodamine 101 sulfonyl chloride (sold under the tradename of TEXAS RED®).

Examples of cyanine dyes include, but are not limited to, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, Cy7, IRDYE680, Alexa Fluor 750, IRDye800CW, ICG.

Examples of fluorescent peptides include GFP (Green Fluorescent Protein) or derivatives of GFP (e.g., EBFP, EBFP2, Azurite, mKalamal, ECFP, Cerulean, CyPet, YFP, Citrine, Venus, YPet).

Fluorescent labels are detected by any suitable method. For example, a fluorescent label may be detected by exciting the fluorochrome with the appropriate wavelength of light and detecting the resulting fluorescence, e.g., by microscopy, visual inspection, via photographic film, by the use of electronic detectors such as charge coupled devices (CCDs), photomultipliers, etc.

In some embodiments, the imaging agent is labeled with a positron-emitting isotope (e.g., ¹⁸F) for positron emission tomography (PET), gamma-ray isotope (e.g., ^(99m)Tc) for single photon emission computed tomography (SPECT), or a paramagnetic molecule or nanoparticle (e.g., Gd³⁺ chelate or coated magnetite nanoparticle) for magnetic resonance imaging (MRI).

In some embodiments, the imaging agent is labeled with: a gadolinium chelate, an iron oxide particle, a super paramagnetic iron oxide particle, an ultra small paramagnetic particle, a manganese chelate or gallium containing agent.

Examples of gadolinium chelates include, but are not limited to diethylene triamine pentaacetic acid (DTPA), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), and 1,4,7-triazacyclononane-N,N′,N″-triacetic acid (NOTA).

In some embodiments, the imaging agent is a near-infrared fluorophore for near-infra red (near-IR) imaging, a luciferase (firefly, bacterial, or coelenterate) or other luminescent molecule for bioluminescence imaging, or a perfluorocarbon-filled vesicle for ultrasound.

In some embodiments, the imaging agent is a nuclear probe. In some embodiments, the imaging agent is a SPECT or PET radionuclide probe. In some embodiments, the radionuclide probe is selected from: a technetium chelate, a copper chelate, a radioactive fluorine, a radioactive iodine, a indiuim chelate.

Examples of Tc chelates include, but are not limited to HYNIC, DTPA, and DOTA.

In some embodiments, the imaging agent contains a radioactive moiety, for example a radioactive isotope such as ²¹¹At, ¹³¹I, ¹²⁵I, ⁹⁰Y, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁵³Sm, ²¹²Bi, ³²P, ⁶⁴Cu radioactive isotopes of Lu, and others.

In some embodiments, a selective delivery molecule according to Formula I comprising an imaging agent is employed in guided surgery. In some embodiments, the selective delivery molecule preferentially localized to cancerous, or other undesirable tissues (i.e. necrotic tissues). In some embodiments, a selective delivery molecule according to Formula I comprising an imaging agent is employed in a guided surgery to remove colorectal cancer. In some embodiments, guided surgery employing the selective delivery molecule allows a surgeon to excise as little healthy (i.e., non-cancerous) tissue as possible. In some embodiments, guided surgery employing the selective delivery molecule allows a surgeon to visualize and excise more cancerous tissue than the surgeon would have been able to excise without the presence of the selective delivery molecule. In some embodiments, the surgery is fluorescence-guided surgery.

Therapeutic Agents

Disclosed herein, in certain embodiments, is the use of a selective delivery molecule disclosed herein for delivering a therapeutic agent to a tissue or a plurality of cells. In some embodiments, the therapeutic agent is an anti-inflammatory agent. In some embodiments, the therapeutic agent is an anti-cancer agent. In some embodiments, the selective delivery molecule is used to treat colorectal cancer.

In some embodiments, a D moiety is independently a therapeutic agent. In some embodiments, the therapeutic agent is selected from: a chemotherapeutic agent, a steroid, an immunotherapeutic agent, a targeted therapy, an anti-inflammatory agent, or a combination thereof.

In some embodiments, the therapeutic agent is a B cell receptor pathway inhibitor. In some embodiments, the therapeutic agent is a CD79A inhibitor, a CD79B inhibitor, a CD19 inhibitor, a Lyn inhibitor, a Syk inhibitor, a PI3K inhibitor, a Blnk inhibitor, a PLCγ inhibitor, a PKCβ inhibitor, or a combination thereof. In some embodiments, the therapeutic agent is an antibody, B cell receptor signaling inhibitor, a PI3K inhibitor, an IAP inhibitor, an mTOR inhibitor, a radioimmunotherapeutic, a DNA damaging agent, a proteosome inhibitor, a histone deacytlase inhibitor, a protein kinase inhibitor, a hedgehog inhibitor, an Hsp90 inhibitor, a telomerase inhibitor, a Jak1/2 inhibitor, a protease inhibitor, a PKC inhibitor, a PARP inhibitor, or a combination thereof. In some embodiments, the therapeutic agent is selected from: chlorambucil, ifosphamide, doxorubicin, mesalazine, thalidomide, lenalidomide, temsirolimus, everolimus, fludarabine, fostamatinib, paclitaxel, docetaxel, ofatumumab, rituximab, dexamethasone, prednisone, CAL-101, ibritumomab, tositumomab, bortezomib, pentostatin, endostatin, bendamustine, chlorambucil, chlormethine, cyclophosphamide, ifosfamide, melphalan, prednimustine, trofosfamide, busulfan, mannosulfan, treosulfan, carboquone, thiotepa, triaziquone, carmustine, fotemustine, lomustine, nimustine, ranimustine, semustine, streptozocin, etoglucid, dacarbazine, mitobronitol, pipobroman, temozolomide, methotrexate, permetrexed, pralatrexate, raltitrexed, cladribine, clofarabine, fludarabine, mercaptopurine, nelarabine, tioguanine, azacitidine, capecitabine, carmofur, cytarabine, decitabine, fluorouracil, gemcitabine, tegafur, vinblastine, vincristine, vindesine, vinflunine, vinorelbine, etoposide, teniposide, demecolcine, docetaxel, paclitaxel, paclitaxel poliglumex, trabectedin, dactinomycin, aclarubicin, daunorubicin, doxorubicin, epirubicin, idarubicin, mitoxantrone, pirarubicin, valrubicin, zorubincin, bleomycin, ixabepilone, mitomycin, plicamycin, carboplatin, cisplatin, oxaliplatin, satraplatin, procarbazine, aminolevulinic acid, efaproxiral, methyl aminolevulinate, porfimer sodium, temoporfin, dasatinib, erlotinib, everolimus, gefitinib, imatinib, lapatinib, nilotinib, pazonanib, sorafenib, sunitinib, temsirolimus, alitretinoin, altretamine, amzacrine, anagrelide, arsenic trioxide, asparaginase, bexarotene, bortezomib, celecoxib, denileukin diftitox, estramustine, hydroxycarbamide, irinotecan, lonidamine, masoprocol, miltefosein, mitoguazone, mitotane, oblimersen, pegaspargase, pentostatin, romidepsin, sitimagene ceradenovec, tiazofurine, topotecan, tretinoin, vorinostat, diethylstilbenol, ethinylestradiol, fosfestrol, polyestradiol phosphate, gestonorone, medroxyprogesterone, megestrol, buserelin, goserelin, leuprorelin, triptorelin, fulvestrant, tamoxifen, toremifene, bicalutamide, flutamide, nilutamide, aminoglutethimide, anastrozole, exemestane, formestane, letrozole, vorozole, abarelix, degarelix, histamine dihydrochloride, mifamurtide, pidotimod, plerixafor, roquinimex, thymopentin, everolimus, gusperimus, leflunomide, mycophenolic acid, sirolimus, ciclosporin, tacrolimus, azathioprine, lenalidomide, methotrexate, thalidomide, iobenguane, ancestim, filgrastim, lenograstim, molgramostim, pegfilgrastim, sargramostim, interferon alfa natural, interferon alfa-2a, interferon alfa-2b, interferon alfacon-1, interferon alfa-n1, interferon beta natural, interferon beta-1a, interferon beta-1b, interferon gamma, peginterferon alfa-2a, peginterferon alfa-2b, aldesleukin, oprelvekin, BCG vaccine, glatiramer acetate, histamine dihydrochloride, immunocyanin, lentinan, melanoma vaccine, mifamurtide, pegademase, pidotimod, plerixafor, poly I:C, poly ICLC, roquinimex, tasonermin, thymopentin, abatacept, abetimus, alefacept, antilymphocyte immunoglobulin (horse), antithymocyte immunoglobulin (rabbit), eculizumab, efalizumab, everolimus, gusperimus, leflunomide, muromab-CD3, mycophenolic acid, natalizumab, sirolimus, adalimumab, afelimomab, certolizumab pegol, etanercept, golimumab, infliximab, anakinra, basiliximab, canakinumab, daclizumab, mepolizumab, rilonacept, tocilizumab, ustekinumab, ciclosporin, tacrolimus, azathioprine, lenalidomide, methotrexate, thalidomide, adalimumab, alemtuzumab, bevacizumab, cetuximab, certolizumab pegol, eculizumab, efalizumab, gemtuzumab, ibritumomab tiuxetan, muromonab-CD3, natalizumab, panitumumab, ranibizumab, rituximab, tositumomab, trastuzumab, catumaxomab, edrecolomab, ofatumumab, muromab-CD3, afelimomab, golimumab, ibritumomab tiuxetan, abagovomab, adecatumumab, alemtuzumab, anti-CD30 monoclonal antibody Xmab2513, anti-MET monoclonal antibody MetMab, apolizumab, apomab, arcitumomab, bispecific antibody 2B1, blinatumomab, brentuximab vedotin, capromab pendetide, cixutumumab, claudiximab, conatumumab, dacetuzumab, denosumab, eculizumab, epratuzumab, epratuzumab, ertumaxomab, etaracizumab, figitumumab, fresolimumab, galiximab, ganitumab, gemtuzumab ozogamicin, glembatumumab, ibritumomab, inotuzumab ozogamicin, ipilimumab, lexatumumab, lintuzumab, lintuzumab, lucatumumab, mapatumumab, matuzumab, milatuzumab, monoclonal antibody CC49, necitumumab, nimotuzumab, ofatumumab, oregovomab, pertuzumab, ramacurimab, ranibizumab, siplizumab, sonepcizumab, tanezumab, tositumomab, trastuzumab, tremelimumab, tucotuzumab celmoleukin, veltuzumab, visilizumab, volociximab, zalutumumab, a syk inhibitor (e.g., R788), enzastaurin, dasatinib, erlotinib, everolimus, gefitinib, imatinib, lapatinib, nilotinib, pazonanib, sorafenib, sunitinib, temsirolimus, an angiogenesis inhibitor (e.g., GT-111, JI-101, R1530), a kinase inhibitors (e.g., AC220, AC480, ACE-041, AMG 900, AP24534, Any-614, AT7519, AT9283, AV-951, axitinib, AZD1152, AZD7762, AZD8055, AZD8931, bafetinib, BAY 73-4506, BGJ398, BGT226, BI 811283, BI6727, BIBF 1120, BIBW 2992, BMS-690154, BMS-777607, BMS-863233, BSK-461364, CAL-101, CEP-11981, CYC116, DCC-2036, dinaciclib, dovitinib lactate, E7050, EMD 1214063, ENMD-2076, fostamatinib disodium, GSK2256098, GSK690693, INCB18424, INNO-406, JNJ-26483327, JX-594, KX2-391, linifanib, LY2603618, MGCD265, MK-0457, MK1496, MLN8054, MLN8237, MP470, NMS-1116354, NMS-1286937, ON 01919.Na, OSI-027, OSI-930, Btk inhibitor, PF-00562271, PF-02341066, PF-03814735, PF-04217903, PF-04554878, PF-04691502, PF-3758309, PHA-739358, PLC3397, progenipoietin, R547, R763, ramucirumab, regorafenib, R05185426, SAR103168, S3333333CH 727965, SGI-1176, SGX523, SNS-314, TAK-593, TAK-901, TKI258, TLN-232, TTP607, XL147, XL228, XL281R05126766, XL418, XL765), an inhibitor of mitogen-activated protein kinase signaling (e.g., U0126, PD98059, PD184352, PD0325901, ARRY-142886, SB239063, SP600125, BAY 43-9006, wortmannin, or LY294002), adriamycin, dactinomycin, bleomycin, vinblastine, cisplatin, acivicin, aclarubicin, acodazole hydrochloride, acronine, adozelesin, aldesleukin, altretamine, ambomycin, ametantrone acetate, aminoglutethimide, amsacrine, anastrozole, anthramycin, asparaginase, asperlin, azacitidine, azetepa, azotomycin, batimastat, benzodepa, bicalutamide, bisantrene hydrochloride, bisnafide dimesylate, bizelesin, bleomycin sulfate, brequinar sodium, bropirimine, busulfan, cactinomycin, calusterone, caracemide, carbetimer, carboplatin, carmustine, carubicin hydrochloride, carzelesin, cedefingol, chlorambucil, cirolemycin, cladribine, crisnatol mesylate, cyclophosphamide, cytarabine, dacarbazine, daunorubicin hydrochloride, decitabine, dexormaplatin, dezaguanine, dezaguanine mesylate, diaziquone, doxorubicin, doxorubicin hydrochloride, droloxifene, droloxifene citrate, dromostanolone propionate, duazomycin, edatrexate, eflornithine hydrochloride, elsamitrucin, enloplatin, enpromate, epipropidine, epirubicin hydrochloride, erbulozole, esorubicin hydrochloride, estramustine, estramustine phosphate sodium, etanidazole, etoposide, etoposide phosphate, etoprine, fadrozole hydrochloride, fazarabine, fenretinide, floxuridine, fludarabine phosphate, fluorouracil, flurocitabine, fosquidone, fostriecin sodium, gemcitabine, gemcitabine hydrochloride, hydroxyurea, idarubicin hydrochloride, ifosfamide, iimofosine, interleukin II (including recombinant interleukin II, or r1L2), interferon alfa-2a, interferon alfa-2b, interferon alfa-n1, interferon alfa-n3, interferon beta-1 a, interferon gamma-1 b, iproplatin, irinotecan hydrochloride, lanreotide acetate, letrozole, leuprolide acetate, liarozole hydrochloride, lometrexol sodium, lomustine, losoxantrone hydrochloride, masoprocol, maytansine, mechlorethamine hydrochloride, megestrol acetate, melengestrol acetate, melphalan, menogaril, mercaptopurine, methotrexate, methotrexate sodium, metoprine, meturedepa, mitindomide, mitocarcin, mitocromin, mitogillin, mitomalcin, mitomycin, mitosper, mitotane, mitoxantrone hydrochloride, mycophenolic acid, nocodazoie, nogalamycin, ormaplatin, oxisuran, pegaspargase, peliomycin, pentamustine, peplomycin sulfate, perfosfamide, pipobroman, piposulfan, piroxantrone hydrochloride, plicamycin, plomestane, porfimer sodium, porfiromycin, prednimustine, procarbazine hydrochloride, puromycin, puromycin hydrochloride, pyrazofurin, riboprine, rogletimide, safingol, safingol hydrochloride, semustine, simtrazene, sparfosate sodium, sparsomycin, spirogermanium hydrochloride, spiromustine, spiroplatin, streptonigrin, streptozocin, sulofenur, talisomycin, tecogalan sodium, tegafur, teloxantrone hydrochloride, temoporfin, teniposide, teroxirone, testolactone, thiamiprine, thioguanine, thiotepa, tiazofurin, tirapazamine, toremifene citrate, trestolone acetate, triciribine phosphate, trimetrexate, trimetrexate glucuronate, triptorelin, tubulozole hydrochloride, uracil mustard, uredepa, vapreotide, verteporfin, vinblastine sulfate, vincristine sulfate, vindesine, vindesine sulfate, vinepidine sulfate, vinglycinate sulfate, vinleurosine sulfate, vinorelbine tartrate, vinrosidine sulfate, vinzolidine sulfate, vorozole, zeniplatin, zinostatin, zorubicin hydrochloride. In some embodiments, the therapeutic agent is selected from: 20-epi-1, 25 dihydroxyvitamin D3, 5-ethynyluracil, abiraterone, aclarubicin, acylfulvene, adecypenol, adozelesin, aldesleukin, ALL-TK antagonists, altretamine, ambamustine, amidox, amifostine, aminolevulinic acid, amrubicin, amsacrine, anagrelide, anastrozole, andrographolide, angiogenesis inhibitors, antagonist D, antagonist G, antarelix, anti-dorsalizing morphogenetic protein-1, antiandrogen, prostatic carcinoma, antiestrogen, antineoplaston, antisense oligonucleotides, aphidicolin glycinate, apoptosis gene modulators, apoptosis regulators, apurinic acid, ara-CDP-DL-PTBA, arginine deaminase, asulacrine, atamestane, atrimustine, axinastatin 1, axinastatin 2, axinastatin 3, azasetron, azatoxin, azatyrosine, baccatin III derivatives, balanol, batimastat, BCR/ABL antagonists, benzochlorins, benzoylstaurosporine, beta lactam derivatives, beta-alethine, betaclamycin B, betulinic acid, bFGF inhibitor, bicalutamide, bisantrene, bisaziridinylspermine, bisnafide, bistratene A, bizelesin, breflate, bropirimine, budotitane, buthionine sulfoximine, calcipotriol, calphostin C, camptothecin derivatives, canarypox IL-2, capecitabine, carboxamide-amino-triazole, carboxyamidotriazole, CaRest M3, CARN 700, cartilage derived inhibitor, carzelesin, casein kinase inhibitors (ICOS), castanospermine, cecropin B, cetrorelix, chlorins, chloroquinoxaline sulfonamide, cicaprost, cis-porphyrin, cladribine, clomifene analogues, clotrimazole, collismycin A, collismycin B, combretastatin A4, combretastatin analogue, conagenin, crambescidin 816, crisnatol, cryptophycin 8, cryptophycin A derivatives, curacin A, cyclopentanthraquinones, cycloplatam, cypemycin, cytarabine ocfosfate, cytolytic factor, cytostatin, dacliximab, decitabine, dehydrodidemnin B, deslorelin, dexamethasone, dexifosfamide, dexrazoxane, dexverapamil, diaziquone, didemnin B, didox, diethylnorspermine, dihydro-5-azacytidine, 9-dioxamycin, diphenyl spiromustine, docosanol, dolasetron, doxifluridine, droloxifene, dronabinol, duocarmycin SA, ebselen, ecomustine, edelfosine, edrecolomab, eflornithine, elemene, emitefur, epirubicin, epristeride, estramustine analogue, estrogen agonists, estrogen antagonists, etanidazole, etoposide phosphate, exemestane, fadrozole, fazarabine, fenretinide, filgrastim, finasteride, flavopiridol, flezelastine, fluasterone, fludarabine, fluorodaunorunicin hydrochloride, forfenimex, formestane, fostriecin, fotemustine, gadolinium texaphyrin, gallium nitrate, galocitabine, ganirelix, gelatinase inhibitors, gemcitabine, glutathione inhibitors, hepsulfam, heregulin, hexamethylene bisacetamide, hypericin, ibandronic acid, idarubicin, idoxifene, idramantone, ilmofosine, ilomastat, imidazoacridones, imiquimod, immunostimulant peptides, insulin-such as for example growth factor-1 receptor inhibitor, interferon agonists, interferons, interleukins, iobenguane, iododoxorubicin, ipomeanol, 4-, iroplact, irsogladine, isobengazole, isohomohalicondrin B, itasetron, jasplakinolide, kahalalide F, lamellarin-N triacetate, lanreotide, leinamycin, lenograstim, lentinan sulfate, leptolstatin, letrozole, leukemia inhibiting factor, leukocyte alpha interferon, leuprolide+estrogen+progesterone, leuprorelin, levamisole, liarozole, linear polyamine analogue, lipophilic disaccharide peptide, lipophilic platinum compounds, lissoclinamide 7, lobaplatin, lombricine, lometrexol, lonidamine, losoxantrone, lovastatin, loxoribine, lurtotecan, lutetium texaphyrin, lysofylline, lytic peptides, maitansine, mannostatin A, marimastat, masoprocol, maspin, matrilysin inhibitors, matrix metalloproteinase inhibitors, menogaril, merbarone, meterelin, methioninase, metoclopramide, MIF inhibitor, mifepristone, miltefosine, mirimostim, mismatched double stranded RNA, mitoguazone, mitolactol, mitomycin analogues, mitonafide, mitotoxin fibroblast growth factor-saporin, mitoxantrone, mofarotene, molgramostim, monoclonal antibody, human chorionic gonadotrophin, monophosphoryl lipid A+myobacterium cell wall sk, mopidamol, multiple drug resistance gene inhibitor, multiple tumor suppressor 1-based therapy, mustard anticancer agent, mycaperoxide B, mycobacterial cell wall extract, myriaporone, N-acetyldinaline, N-substituted benzamides, nafarelin, nagrestip, naloxone+pentazocine, napavin, naphterpin, nartograstim, nedaplatin, nemorubicin, neridronic acid, neutral endopeptidase, nilutamide, nisamycin, nitric oxide modulators, nitroxide antioxidant, nitrullyn, O6-benzylguanine, octreotide, okicenone, oligonucleotides, onapristone, ondansetron, ondansetron, oracin, oral cytokine inducer, ormaplatin, osaterone, oxaliplatin, oxaunomycin, palauamine, palmitoylrhizoxin, pamidronic acid, panaxytriol, panomifene, parabactin, pazelliptine, pegaspargase, peldesine, pentosan polysulfate sodium, pentostatin, pentrozole, perflubron, perfosfamide, perillyl alcohol, phenazinomycin, phenylacetate, phosphatase inhibitors, picibanil, pilocarpine hydrochloride, pirarubicin, piritrexim, placetin A, placetin B, plasminogen activator inhibitor, platinum complex, platinum compounds, platinum-triamine complex, porfimer sodium, porfiromycin, prednisone, propyl bis-acridone, prostaglandin J2, proteasome inhibitors, protein A-based immune modulator, protein kinase C inhibitor, protein kinase C inhibitors, microalgal, protein tyrosine phosphatase inhibitors, purine nucleoside phosphorylase inhibitors, purpurins, pyrazoloacridine, pyridoxylated hemoglobin polyoxyethylerie conjugate, raf antagonists, raltitrexed, ramosetron, ras farnesyl protein transferase inhibitors, ras inhibitors, ras-GAP inhibitor, retelliptine demethylated, rhenium Re 186 etidronate, rhizoxin, ribozymes, RH retinamide, rogletimide, rohitukine, romurtide, roquinimex, rubiginone B1, ruboxyl, safingol, saintopin, SarCNU, sarcophytol A, sargramostim, Sdi 1 mimetics, semustine, senescence derived inhibitor 1, sense oligonucleotides, signal transduction inhibitors, signal transduction modulators, single chain antigen-binding protein, sizofiran, sobuzoxane, sodium borocaptate, sodium phenylacetate, solverol, somatomedin binding protein, sonermin, sparfosic acid, spicamycin D, spiromustine, splenopentin, spongistatin 1, squalamine, stem cell inhibitor, stem-cell division inhibitors, stipiamide, stromelysin inhibitors, sulfinosine, superactive vasoactive intestinal peptide antagonist, suradista, suramin, swainsonine, synthetic glycosaminoglycans, tallimustine, tamoxifen methiodide, tauromustine, tazarotene, tecogalan sodium, tegafur, tellurapyrylium, telomerase inhibitors, temoporfin, temozolomide, teniposide, tetrachlorodecaoxide, tetrazomine, thaliblastine, thiocoraline, thrombopoietin, thrombopoietin mimetic, thymalfasin, thymopoietin receptor agonist, thymotrinan, thyroid stimulating hormone, tin ethyl etiopurpurin, tirapazamine, titanocene bichloride, topsentin, toremifene, totipotent stem cell factor, translation inhibitors, tretinoin, triacetyluridine, triciribine, trimetrexate, triptorelin, tropisetron, turosteride, tyrosine kinase inhibitors, tyrphostins, UBC inhibitors, ubenimex, urogenital sinus-derived growth inhibitory factor, urokinase receptor antagonists, vapreotide, variolin B, vector system, erythrocyte gene therapy, velaresol, veramine, verdins, verteporfin, vinorelbine, vinxaltine, vitaxin, vorozole, zanoterone, zeniplatin, zilascorb, zinostatin stimalamer, mechloroethamine, cyclophosphamide, chlorambucil, busulfan, carmustine, lomusitne, decarbazine, methotrexate, cytarabine, mercaptopurine, thioguanine, pentostatin, mechloroethamine, cyclophosphamide, chlorambucil, meiphalan, ethylenimine, methylmelamine, hexamethlymelamine, thiotepa, busulfan, carmustine, lomusitne, semustine, streptozocin, decarbazine, fluorouracil, floxouridine, cytarabine, mercaptopurine, thioguanine, pentostatin, erbulozole (also known as R-55104), Dolastatin 10 (also known as DLS-10 and NSC-376128), Mivobulin isethionate (also known as CI-980), Vincristine, NSC-639829, Discodermolide (also known as NVP-XX-A-296), ABT-751 (Abbott, also known as E-7010), Altorhyrtins (such as Altorhyrtin A and Altorhyrtin C), Spongistatins (such as Spongistatin 1, Spongistatin 2, Spongistatin 3, Spongistatin 4, Spongistatin 5, Spongistatin 6, Spongistatin 7, Spongistatin 8, and Spongistatin 9), Cemadotin hydrochloride (also known as LU-103793 and NSC-D-669356), Epothilones (such as Epothilone A, Epothilone B, Epothilone C (also known as desoxyepothilone A or dEpoA), Epothilone D (also referred to as KOS-862, dEpoB, and desoxyepothilone B), Epothilone E, Epothilone F, Epothilone B N-oxide, Epothilone A N-oxide, 16-aza-epothilone B, 21-aminoepothilone B (also known as BMS-310705), 21-hydroxyepothilone D (also known as Desoxyepothilone F and dEpoF), 26-fluoroepothilone), Auristatin PE (also known as NSC-654663), Soblidotin (also known as TZT-1027), LS-4559-P (Pharmacia, also known as LS-4577), LS-4578 (Pharmacia, also known as LS-477-P), LS-4477 (Pharmacia), LS-4559 (Pharmacia), RPR-112378 (Aventis), Vincristine sulfate, DZ-3358 (Daiichi), FR-182877 (Fujisawa, also known as WS-9885B), GS-164 (Takeda), GS-198 (Takeda), KAR-2 (Hungarian Academy of Sciences), BSF-223651 (BASF, also known as ILX-651 and LU-223651), SAH-49960 (Lilly/Novartis), SDZ-268970 (Lilly/Novartis), AM-97 (Armad/Kyowa Hakko), AM-132 (Armad), AM-138 (Armad/Kyowa Hakko), IDN-5005 (Indena), Cryptophycin 52 (also known as LY-355703), AC-7739 (Ajinomoto, also known as AVE-8063A and CS-39.HCl), AC-7700 (Ajinomoto, also known as AVE-8062, AVE-8062A, CS-39-L-Ser.HCl, and RPR-258062A), Vitilevuamide, Tubulysin A, Canadensol, Centaureidin (also known as NSC-106969), T-138067 (Tularik, also known as T-67, TL-138067 and TI-138067), COBRA-1 (Parker Hughes Institute, also known as DDE-261 and WHI-261), H10 (Kansas State University), H16 (Kansas State University), Oncocidin A1 (also known as BTO-956 and DIME), DDE-313 (Parker Hughes Institute), Fijianolide B, Laulimalide, SPA-2 (Parker Hughes Institute), SPA-1 (Parker Hughes Institute, also known as SPIKET-P), 3-IAABU (Cytoskeleton/Mt. Sinai School of Medicine, also known as MF-569), Narcosine (also known as NSC-5366), Nascapine, D-24851 (Asta Medica), A-105972 (Abbott), Hemiasterlin, 3-BAABU (Cytoskeleton/Mt. Sinai School of Medicine, also known as MF-191), TMPN (Arizona State University), Vanadocene acetylacetonate, T-138026 (Tularik), Monsatrol, Inanocine (also known as NSC-698666), 3-1AABE (Cytoskeleton/Mt. Sinai School of Medicine), A-204197 (Abbott), T-607 (Tuiarik, also known as T-900607), RPR-115781 (Aventis), Eleutherobins (such as Desmethyleleutherobin, Desaetyleleutherobin, lsoeleutherobin A, and Z-Eleutherobin), Caribaeoside, Caribaeolin, Halichondrin B, D-64131 (Asta Medica), D-68144 (Asta Medica), Diazonamide A, A-293620 (Abbott), NPI-2350 (Nereus), Taccalonolide A, TUB-245 (Aventis), A-259754 (Abbott), Diozostatin, (−)-Phenylahistin (also known as NSCL-96F037), D-68838 (Asta Medica), D-68836 (Asta Medica), Myoseverin B, D-43411 (Zentaris, also known as D-81862), A-289099 (Abbott), A-318315 (Abbott), HTI-286 (also known as SPA-110, trifluoroacetate salt) (Wyeth), D-82317 (Zentaris), D-82318 (Zentaris), SC-12983 (NCI), Resverastatin phosphate sodium, BPR-OY-007 (National Health Research Institutes), and SSR-250411 (Sanofi).

In some embodiments, the therapeutic agent is an anti-inflammatory agent. In some embodiments, the therapeutic agent is an anti-TNF agent, an IL-1 receptor antagonist, an IL-2 receptor antagonist, a cytotoxic agent, an immunomodulatory agent, an antibiotic, a T-cell co-stimulatory blocker, a B cell depleting agent, an immunosuppressive agent, an alkylating agent, an anti-metabolite, a plant alkaloid, a terpenoids, a topoisomerase inhibitor, an antitumour antibiotic, an antibody, a hormonal therapy, an anti-diabetes agent, a leukotriene inhibitor, or combinations thereof. In some embodiments, the therapeutic agent is selected from: alefacept, efalizumab, methotrexate, acitretin, isotretinoin, hydroxyurea, mycophenolate mofetil, sulfasalazine, 6-Thioguanine, Dovonex, Taclonex, betamethasone, tazarotene, hydroxychloroquine, etanercept, adalimumab, infliximab, abatacept, rituximab, tratuzumab, Anti-CD45 monoclonal antibody AHN-12 (NCI), Iodine-131 Anti-B1 Antibody (Corixa Corp.), anti-CD66 monoclonal antibody BW 250/183 (NCI, Southampton General Hospital), anti-CD45 monoclonal antibody (NCI, Baylor College of Medicine), antibody anti-anb3 integrin (NCI), BIW-8962 (BioWa Inc.), Antibody BC8 (NCI), antibody muJ591 (NCI), indium In 111 monoclonal antibody MN-14 (NCI), yttrium Y 90 monoclonal antibody MN-14 (NCI), F105 Monoclonal Antibody (NIAID), Monoclonal Antibody RAV12 (Raven Biotechnologies), CAT-192 (Human Anti-TGF-Betal Monoclonal Antibody, Genzyme), antibody 3F8 (NCI), 177Lu-J591 (Weill Medical College of Cornell University), TB-403 (Biolnvent International AB), anakinra, azathioprine, cyclophosphamide, cyclosporine A, leflunomide, d-penicillamine, amitriptyline, or nortriptyline, chlorambucil, nitrogen mustard, prasterone, LJP 394 (abetimus sodium), LJP 1082 (La Jolla Pharmaceutical), eculizumab, belibumab, rhuCD40L (NIAID), epratuzumab, sirolimus, tacrolimus, pimecrolimus, thalidomide, antithymocyte globulin-equine (Atgam, Pharmacia Upjohn), antithymocyte globulin-rabbit (Thymoglobulin, Genzyme), Muromonab-CD3 (FDA Office of Orphan Products Development), basiliximab, daclizumab, riluzole, cladribine, natalizumab, interferon beta-1b, interferon beta-1a, tizanidine, baclofen, mesalazine, asacol, pentasa, mesalamine, balsalazide, olsalazine, 6-mercaptopurine, AIN457 (Anti IL-17 Monoclonal Antibody, Novartis), theophylline, D2E7 (a human anti-TNF mAb from Knoll Pharmaceuticals), Mepolizumab (Anti-IL-5 antibody, SB 240563), Canakinumab (Anti-IL-1 Beta Antibody, NIAMS), Anti-IL-2 Receptor Antibody (Daclizumab, NHLBI), CNTO 328 (Anti IL-6 Monoclonal Antibody, Centocor), ACZ885 (fully human anti-interleukin-lbeta monoclonal antibody, Novartis), CNTO 1275 (Fully Human Anti-IL-12 Monoclonal Antibody, Centocor), (3S)-N-hydroxy-4-({4-[(4-hydroxy-2-butynyl)oxy]phenyl}sulfonyl)-2,2-dimethyl-3-thiomorpholine carboxamide (apratastat), golimumab (CNTO 148), Onercept, BG9924 (Biogen Idec), Certolizumab Pegol (CDP870, UCB Pharma), AZD9056 (AstraZeneca), AZD5069 (AstraZeneca), AZD9668 (AstraZeneca), AZD7928 (AstraZeneca), AZD2914 (AstraZeneca), AZD6067 (AstraZeneca), AZD3342 (AstraZeneca), AZD8309 (AstraZeneca),), [(1R)-3-methyl-1-({(2S)-3-phenyl-2-[(pyrazin-2-ylcarbonyl)amino]propanoyl}amino)butyl]boronic acid (Bortezomib), AMG-714, (Anti-IL 15 Human Monoclonal Antibody, Amgen), ABT-874 (Anti IL-12 monoclonal antibody, Abbott Labs), MRA(Tocilizumab, an Anti IL-6 Receptor Monoclonal Antibody, Chugai Pharmaceutical), CAT-354 (a human anti-interleukin-13 monoclonal antibody, Cambridge Antibody Technology, MedImmune), aspirin, salicylic acid, gentisic acid, choline magnesium salicylate, choline salicylate, choline magnesium salicylate, choline salicylate, magnesium salicylate, sodium salicylate, diflunisal, carprofen, fenoprofen, fenoprofen calcium, flurobiprofen, ibuprofen, ketoprofen, nabutone, ketolorac, ketorolac tromethamine, naproxen, oxaprozin, diclofenac, etodolac, indomethacin, sulindac, tolmetin, meclofenamate, meclofenamate sodium, mefenamic acid, piroxicam, meloxicam, celecoxib, rofecoxib, valdecoxib, parecoxib, etoricoxib, lumiracoxib, CS-502 (Sankyo), JTE-522 (Japan Tobacco Inc.), L-745,337 (Almirall), NS398 (Sigma), betamethasone (Celestone), prednisone (Deltasone), alclometasone, aldosterone, amcinonide, beclometasone, betamethasone, budesonide, ciclesonide, clobetasol, clobetasone, clocortolone, cloprednol, cortisone, cortivazol, deflazacort, deoxycorticosterone, desonide, desoximetasone, desoxycortone, dexamethasone, diflorasone, diflucortolone, difluprednate, fluclorolone, fludrocortisone, fludroxycortide, flumetasone, flunisolide, fluocinolone acetonide, fluocinonide, fluocortin, fluocortolone, fluorometholone, fluperolone, fluprednidene, fluticasone, formocortal, formoterol, halcinonide, halometasone, hydrocortisone, hydrocortisone aceponate, hydrocortisone buteprate, hydrocortisone butyrate, loteprednol, medrysone, meprednisone, methylprednisolone, methylprednisolone aceponate, mometasone furoate, paramethasone, prednicarbate, prednisone, rimexolone, tixocortol, triamcinolone, ulobetasol, Pioglitazone, Rosiglitazone, Glimepiride, Glyburide, Chlorpropamide, Glipizide, Tolbutamide, Tolazamide, Glucophage, Metformin, (glyburide+metformin), Rosiglitazone+metformin, (Rosiglitazone+glimepiride), Exenatide, Insulin, Sitagliptin, (glipizide and metformin), Repaglinide, Acarbose, Nateglinide, Orlistat, cisplatin; carboplatin; oxaliplatin; mechlorethamine; cyclophosphamide; chlorambucil; vincristine; vinblastine; vinorelbine; vindesine; mercaptopurine; fludarabine; pentostatin; cladribine; 5-fluorouracil (5FU); floxuridine (FUDR); cytosine arabinoside; trimethoprim; pyrimethamine; pemetrexed; paclitaxel; docetaxel; etoposide; teniposide; irinotecan; topotecan; amsacrine; etoposide; etoposide phosphate; teniposide; dactinomycin; doxorubicin; daunorubicin; valrubicine; idarubicine; epirubicin; bleomycin; plicamycin; mitomycin; finasteride; goserelin; aminoglutethimide; anastrozole; letrozole; vorozole; exemestane; 4-androstene-3,6,17-trione (“6-OXO”; 1,4,6-androstatrien-3,17-dione (ATD); formestane; testolactone; fadrozole; A-81834 (3-(3-(1,1-dimethylethylthio-5-(quinoline-2-ylmethoxy)-1-(4-chloromethylphenyl)indole-2-yl)-2,2-dimethylpropionaldehyde oxime-O-2-acetic acid; AME103 (Amira); AME803 (Amira); atreleuton; BAY-x-1005 ((R)-(+)-alpha-cyclopentyl-4-(2-quinolinylmethoxy)-Benzeneacetic acid); CJ-13610 (4-(3-(4-(2-Methyl-imidazol-1-yl)-phenylsulfanyl)-phenyl)-tetrahydro-pyran-4-carboxylic acid amide); DG-031 (DeCode); DG-051 (DeCode); MK886 (1-[(4-chlorophenyl)methyl]3-[(1,1-dimethylethyl)thio]-α,α-dimethyl-5-(1-methylethyl)-1H-indole-2-propanoic acid, sodium salt); MK591 (3-(1-4[(4-chlorophenyl)methyl]-3-[(t-butylthio)-5-((2-quinoly)methoxy)-1H-indole-21-, dimehtylpropanoic acid); RP64966 ([4-[5-(3-Phenyl-propyl)thiophen-2-yl]butoxy]acetic acid); SA6541 ((R)-S-[[4-(dimethylamino)phenyl]methyl]-N-(3-mercapto-2methyl-1-oxopropyl-L-cycteine); SC-56938 (ethyl-1-[2-[4-(phenylmethyl)phenoxy]ethyl]-4-piperidine-carboxylate); VIA-2291 (Via Pharmaceuticals); WY-47,288 (2-[(1-naphthalenyloxy)methyl]quinoline); zileuton; ZD-2138 (6-((3-fluoro-5-(tetrahydro-4-methoxy-2H-pyran-4yl)phenoxy)methyl)-1-methyl-2(1H)-quinlolinone); doxycycline; or combinations thereof.

Macromolecular Carriers

The term “carrier” means an inert molecule that modulates plasma half-life, solubility, or bio-distribution. In some embodiments, a carrier modulates plasma half-life of a selective delivery molecule disclosed herein. In some embodiments, a carrier modulates solubility of a selective delivery molecule disclosed herein. In some embodiments, a carrier modulates bio-distribution of a selective delivery molecule disclosed herein.

In some embodiments, a carrier decreases uptake of a selective delivery molecule by non-target cells or tissues. In some embodiments, a carrier decreases uptake of a selective delivery molecule into cartilage. In some embodiments, a carrier decreases uptake of a selective delivery molecule into joints relative to target tissue.

In some embodiments, a carrier increases uptake of a selective delivery molecule by target cells or tissues. In some embodiments, a carrier decreases uptake of a selective delivery molecule into the liver relative to target tissue. In some embodiments, a carrier decreases uptake of a selective delivery molecule into kidneys. In some embodiments, a carrier enhances uptake into cancer tissue. In some embodiments, a carrier enhances uptake into lymphatic channels and/or lymph nodes.

In some embodiments, a carrier increases plasma half-life by reducing glomerular filtration. In some embodiments, a carrier modulates plasma half-life by increasing or decreases metabolism or protease degradation. In some embodiments, a carrier increases tumor uptake due to enhanced permeability and retention (EPR) of tumor vasculature. In some embodiments, a carrier increases the aqueous solubility of selective delivery molecule.

In some embodiments, any M is independently directly or indirectly (e.g., via c_(M)) bound to A, B, or X. In some embodiments, any M is independently bound to A at the n-terminal poly glutamate. In some embodiments, any M is independently bound to A (or, the n-terminal poly glutamate) by a covalent linkage. In some embodiments, any M is independently bound to B at the c-terminal polyarginine. In some embodiments, any M is independently bound to B (or, the c-terminal polyarginine) by a covalent linkage. In some embodiments, any M is independently directly or indirectly bound to linkers between X and A, X and B, B and C/N terminus, and A and C/N terminus. In some embodiments, the covalent linkage comprises an ether bond, thioether bond, amine bond, amide bond, oxime bond, carbon-carbon bond, carbon-nitrogen bond, carbon-oxygen bond, or carbon-sulfur bond.

In some embodiments, M is selected from a protein, a synthetic or natural polymer, or a dendrimer. In some embodiments, M is selected from dextran, a PEG polymer (e.g., PEG 5 kDa, PEG 12 kDa, PEG 20 kDa, PEG 30 kDa, and PEG40 kDa), albumin, or a combination thereof. In some embodiments, M is a PEG polymer.

In some embodiments, the size of M is between 50 and 70 kD.

In some embodiments, the selective delivery molecule is conjugated to albumin. In certain instances, albumin is excluded from the glomerular filtrate under normal physiological conditions. In some embodiments, the selective delivery molecule comprises a reactive group such as maleimide that can form a covalent conjugate with albumin A selective delivery molecule comprising albumin results in enhanced accumulation of cleaved selective delivery molecules in tumors in a cleavage dependent manner. In some embodiments, albumin conjugates have good pharmacokinetic properties.

In some embodiments, the selective delivery molecule is conjugated to a PEG polymer. In some embodiments, the selective delivery molecule is conjugated to a PEG 5 kDa polymer. In some embodiments, the selective delivery molecule is conjugated to a PEG 12 kDa polymer. In some embodiments, selective delivery molecule is conjugated to a PEG 20 kDa polymer. In some embodiments, 30 kD PEG conjugates had a longer half-life as compared to free peptides. In some embodiments, selective delivery molecules are conjugated to 20-40 kD PEG polymer which has hepatic and renal clearance.

In some embodiments, the selective delivery molecule is conjugated to a dextran. In some embodiments, the selective delivery molecule is conjugated to a 70 kDa dextran. In some embodiments, dextran conjugates, being a mixture of molecular weights, are difficult to synthesize and purify reproducibly.

In some embodiments, the selective delivery molecule is conjugated to streptavidin.

In some embodiments, the selective delivery molecule is conjugated to a fifth generation PAMAM dendrimer.

In some embodiments, a carrier is capped. In some embodiments, capping a carrier improves the pharmacokinetics and reduces cytotoxicity of a carrier by adding hydrophilicity. In some embodiments, the cap is selected from: Acetyl, succinyl, 3-hydroxypropionyl, 2-sulfobenzoyl, glycidyl, PEG-2, PEG-4, PEG-8 and PEG-12.

Portion X (Linkers)

In some embodiments, a linker consisting of one or more amino acids is used to join peptide sequence A (i.e., the sequence designed to inhibit the delivery action of peptide B) and peptide sequence B. Generally the peptide linker will have no specific biological activity other than to join the molecules or to preserve some minimum distance or other spatial relationship between them. However, the constituent amino acids of the linker may be selected to influence some property of the molecule such as the folding, net charge, or hydrophobicity.

In live cells, an intact selective delivery molecule disclosed herein may not be able to enter the cell because of the presence of portion A. Thus, a strictly intracellular process for cleaving X would be ineffective to cleave X in healthy cells since portion A, preventing uptake into cells, would not be effectively cleaved by intracellular enzymes in healthy cells since it would not be taken up and would not gain access to such intracellular enzymes. However, where a cell is injured or diseased (e.g., cancerous cells, hypoxic cells, ischemic cells, apoptotic cells, necrotic cells) such intracellular enzymes leak out of the cell and cleavage of A would occur, allowing entry of portion B and/or cargo into the cell, effecting targeted delivery of portion B and/or cargo D to neighboring cells. In some embodiments, X is cleaved in the extracellular space.

In some embodiments, the fact that capillaries are often leaky around tumors and other trauma sites enhances the ability of high molecular weight molecules (e.g., molecular weight of about 30 kDa or more) to reach the interstitial compartment. In some embodiments, X linkercells that do not express the relevant protease but that are immediately adjacent to expressing cells pick up cargo from a selective delivery molecule because linkage of a X linker is typically extracellular. In some embodiments, such bystander targeting is beneficial in the treatment of tumors because of the heterogeneity of cell phenotypes and the wish to eliminate as high a percentage of suspicious cells as possible.

In some embodiments, X is a cleavable linker.

In some embodiments, the linker is flexible. In some embodiments, the linker is rigid.

In some embodiments, the linker comprises a linear structure. In some embodiments, the linker comprises a non-linear structure. In some embodiments, the linker comprises a branched structure. In some embodiments, the linker comprises a cyclic structure.

In some embodiments, X is about 5 to about 30 atoms in length. In some embodiments, X is about 6 atoms in length. In some embodiments, X is about 8 atoms in length. In some embodiments, X is about 10 atoms in length. In some embodiments, X is about 12 atoms in length. In some embodiments, X is about 14 atoms in length. In some embodiments, X is about 16 atoms in length. In some embodiments, X is about 18 atoms in length. In some embodiments, X is about 20 atoms in length. In some embodiments, X is about 25 atoms in length. In some embodiments, X is about 30 atoms in length.

In some embodiments, the linker binds peptide portion A (i.e., the peptide sequence which prevents cellular uptake) to peptide portion B (i.e., the delivery sequence) by a covalent linkage. In some embodiments, the covalent linkage comprises an ether bond, thioether bond, amine bond, amide bond, oxime bond, hydrazone bond, carbon-carbon bond, carbon-nitrogen bond, carbon-oxygen bond, or carbon-sulfur bond.

In some embodiments, X comprises a peptide linkage. The peptide linkage comprises L-amino acids and/or D-amino acids. In embodiments of the invention, D-amino acids are preferred in order to minimize immunogenicity and nonspecific cleavage by background peptidases or proteases. Cellular uptake of oligo-D-arginine sequences is known to be as good as or better than that of oligo-L-arginines.

In some embodiments, a X linker is designed for cleavage in the presence of particular conditions or in a particular environment. In preferred embodiments, a X linker is cleavable under physiological conditions. Cleavage of such a X linker may, for example, be enhanced or may be affected by particular pathological signals or a particular environment related to cells in which cargo delivery is desired. The design of a X linker for cleavage by specific conditions, such as by a specific enzyme, allows the targeting of cellular uptake to a specific location where such conditions obtain. Thus, one important way that selective delivery molecules provide specific targeting of cellular uptake to desired cells, tissues, or regions is by the design of the linker portion X to be cleaved by conditions near such targeted cells, tissues, or regions.

In some embodiments, X is a pH-sensitive linker. In some embodiments, X is cleaved under basic pH conditions. In some embodiments, X is cleaved under acidic pH conditions. In some embodiments, X is cleaved by a protease, a matrix metalloproteinase, or a combination thereof. In some embodiments, X is cleaved by a reducing agent.

In some embodiments, X is cleaved by an MMP. The hydrolytic activity of matrix metalloproteinases (MMPs) has been implicated in the invasive migration of metastatic tumor cells. In certain instances, MMPs are found near sites of inflammation. In certain instances, MMPs are found near sites of stroke (i.e., a disorder characterized by brain damage following a decrease in blood flow). Thus, uptake of molecules having features of the invention are able to direct cellular uptake of cargo (at least one D moiety) to specific cells, tissues, or regions having active MMPs in the extracellular environment. In some embodiments, a X linker that includes the amino-acid sequences PLG-C(Me)-AG (SEQ ID NO: 1), PLGLAG (SEQ ID NO: 2) which are cleaved by the metalloproteinase enzymes MMP-2, MMP-9, or MMP-7 (MMPs involved in cancer and inflammation).

In some embodiments, X is cleaved by proteolytic enzymes or reducing environment, as may be found near cancerous cells. Such an environment, or such enzymes, are typically not found near normal cells.

In some embodiments, X is cleaved by serine proteases including but not limited to thrombin.

In some embodiments, X is cleaved in or near tissues suffering from hypoxia. In some embodiments, cleavage in or near hypoxic tissues enables targeting of cancer cells and cancerous tissues, infarct regions, and other hypoxic regions. In some embodiments, X comprises a disulfide bond. In some embodiments, a linker comprising a disulfide bond is preferentially cleaved in hypoxic regions and so targets cargo delivery to cells in such a region. Hypoxia is thought to cause cancer cells to become more resistant to radiation and chemotherapy, and also to initiate angiogenesis. In a hypoxic environment in the presence of, for example, leaky or necrotic cells, free thiols and other reducing agents become available extracellularly, while the O₂ that normally keeps the extracellular environment oxidizing is by definition depleted. In some embodiments, this shift in the redox balance promotes reduction and cleavage of a disulfide bond within a X linker. In addition to disulfide linkages which take advantage of thiol-disulfide equilibria, linkages including quinones that fall apart when reduced to hydroquinones are used in a X linker designed to be cleaved in a hypoxic environment.

In some embodiments, X is cleaved in a necrotic environment. Necrosis often leads to the release of enzymes or other cell contents that may be used to trigger cleavage of a X linker. In some embodiments, cleavage of X by necrotic enzymes (e.g., by calpains) allows cargo to be taken up by diseased cells and by neighboring cells that had not yet become fully leaky.

In some embodiments, X is an acid-labile linker. In some embodiments, X comprises an acetal or vinyl ether linkage. Acidosis is observed in sites of damaged or hypoxic tissue, due to the Warburg shift from oxidative phosphorylation to anaerobic glycolysis and lactic acid production. In some embodiments, acidosis is used as a trigger of cargo uptake by replacing some of the arginines within B by histidines, which only become cationic below pH 7.

It will be understood that a linker disclosed herein may include non-standard amino acids, such as, for example, hydroxylysine, desmosine, isodesmosine, or other non-standard amino acids. A linker disclosed herein may include modified amino acids, including post-translationally modified amino acids such as, for example, methylated amino acids (e.g., methyl histidine, methylated forms of lysine, etc.), acetylated amino acids, amidated amino acids, formylated amino acids, hydroxylated amino acids, phosphorylated amino acids, or other modified amino acids. A linker disclosed herein may also include peptide mimetic moieties, including portions linked by non-peptide bonds and amino acids linked by or to non-amino acid portions.

In some embodiments, the linker X comprises an amino acid sequence selected from: PLGLAG (SEQ ID NO: 2), PLG-C(me)-AG (SEQ ID NO: 1), RPLALWRS (SEQ ID NO: 7), ESPAYYTA (SEQ ID NO: 8), DPRSFL (SEQ ID NO: 9), PPRSFL (SEQ ID NO: 10), RLQLKL (SEQ ID NO: 11), and RLQLK(Ac) (SEQ ID NO: 12). In some embodiments, the linker X comprises the amino acid sequence PLGLAG (SEQ ID NO: 2). In some embodiments, the linker X comprises the amino acid sequence PLG-C(me)-AG (SEQ ID NO: 1). In some embodiments, the linker X comprises the amino acid sequence PLGxAG (SEQ ID NO: 27), wherein x is any amino acid (naturally-occuring or non-naturally occurring). In some embodiments, the linker X comprises the amino acid sequence RPLALWRS (SEQ ID NO: 7). In some embodiments, the linker X comprises the amino acid sequence ESPAYYTA (SEQ ID NO: 8). In some embodiments, the linker X comprises the amino acid sequence DPRSFL (SEQ ID NO: 9). In some embodiments, the linker X comprises the amino acid sequence PPRSFL (SEQ ID NO: 10). In some embodiments, the linker X comprises the amino acid sequence RLQLKL (SEQ ID NO: 11). In some embodiments, the linker X comprises the amino acid sequence RLQLK(Ac) (SEQ ID NO: 12).

In some embodiments, the linker X comprises a peptide selected from: PR(S/T)(L/I)(S/T), where the letters in parentheses indicate that either one of the indicated amino acids may be at that position in the sequence); GGAANLVRGG (SEQ ID NO: 28); SGRIGFLRTA (SEQ ID NO: 29); SGRSA (SEQ ID NO: 30); GFLG (SEQ ID NO: 31); ALAL (SEQ ID NO: 32); FK; PIC(Et)F-F (SEQ ID NO: 33), where C(Et) indicates S-ethylcysteine (a cysteine with an ethyl group attached to the thiol) and the “-” indicates the typical cleavage site in this and subsequent sequences); GGPRGLPG (SEQ ID NO: 34); HSSKLQ (SEQ ID NO: 35); LVLA-SSSFGY (SEQ ID NO: 36); GVSQNY-PIVG (SEQ ID NO: 37); GVVQA-SCRLA (SEQ ID NO: 38); f(Pip)R-S, where “f” indicates D-phenylalanine and “Pip” indicates piperidine-2-carboxylic acid (pipecolinic acid, a proline analog having a six-membered ring); DEVD (SEQ ID NO: 39); GWEHDG (SEQ ID NO: 40); RPLALWRS (SEQ ID NO: 7), or a combination thereof.

In some embodiments, X is cleaved under hypoxic conditions. In some embodiments, X comprises a disulfide linkage. In some embodiments, X comprises a quinine

In some embodiments, X is cleaved under necrotic conditions. In some embodiments, X comprises a molecule cleavable by a calpain.

In some embodiments, X comprises 6-aminohexanoyl, 5-(amino)-3-oxapentanoyl, or a combination thereof. In some embodiments, X comprises a disulfide linkage.

In some embodiments, the linker is an alkyl. In some embodiments, the linker is hetero alkyl.

In some embodiments, the linker is an alkylene. In some embodiments, the linker is an alkenylene. In some embodiments, the linker is an alkynylene. In some embodiments, the linker is a heteroalkylene.

An “alkyl” group refers to an aliphatic hydrocarbon group. The alkyl moiety may be a saturated alkyl or an unsaturated alkyl. Depending on the structure, an alkyl group can be a monoradical or a diradical (i.e., an alkylene group).

The “alkyl” moiety may have 1 to 10 carbon atoms (whenever it appears herein, a numerical range such as “1 to 10” refers to each integer in the given range; e.g., “1 to 10 carbon atoms” means that the alkyl group may consist of 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc., up to and including 10 carbon atoms, although the present definition also covers the occurrence of the term “alkyl” where no numerical range is designated). The alkyl group could also be a “lower alkyl” having 1 to 6 carbon atoms. The alkyl group of the compounds described herein may be designated as “C1-C4 alkyl” or similar designations. By way of example only, “C1-C4 alkyl” indicates that there are one to four carbon atoms in the alkyl chain, i.e., the alkyl chain is selected from: methyl, ethyl, propyl, iso-propyl, n-butyl, iso-butyl, sec-butyl, and t-butyl. Typical alkyl groups include, but are in no way limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tertiary butyl, pentyl, hexyl, ethenyl, propenyl, butenyl, and the like.

In some embodiments, the linker comprises a ring structure (e.g., an aryl). As used herein, the term “ring” refers to any covalently closed structure. Rings include, for example, carbocycles (e.g., aryls and cycloalkyls), heterocycles (e.g., heteroaryls and non-aromatic heterocycles), aromatics (e.g. aryls and heteroaryls), and non-aromatics (e.g., cycloalkyls and non-aromatic heterocycles). Rings can be optionally substituted. Rings can be monocyclic or polycyclic.

As used herein, the term “aryl” refers to an aromatic ring wherein each of the atoms forming the ring is a carbon atom. Aryl rings can be formed by five, six, seven, eight, nine, or more than nine carbon atoms. Aryl groups can be optionally substituted. Examples of aryl groups include, but are not limited to phenyl, naphthalenyl, phenanthrenyl, anthracenyl, fluorenyl, and indenyl. Depending on the structure, an aryl group can be a monoradical or a diradical (i.e., an arylene group).

The term “cycloalkyl” refers to a monocyclic or polycyclic non-aromatic radical, wherein each of the atoms forming the ring (i.e. skeletal atoms) is a carbon atom. Cycloalkyls may be saturated, or partially unsaturated. Cycloalkyl groups include groups having from 3 to 10 ring atoms. Cycloalkyls include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl.

In some embodiments, the ring is a cycloalkane. In some embodiments, the ring is a cycloalkene.

In some embodiments, the ring is an aromatic ring. The term “aromatic” refers to a planar ring having a delocalized i-electron system containing 4n+2 π electrons, where n is an integer. Aromatic rings can be formed from five, six, seven, eight, nine, or more than nine atoms. Aromatics can be optionally substituted. The term “aromatic” includes both carbocyclic aryl (e.g., phenyl) and heterocyclic aryl (or “heteroaryl” or “heteroaromatic”) groups (e.g., pyridine). The term includes monocyclic or fused-ring polycyclic (i.e., rings which share adjacent pairs of carbon atoms) groups.

In some embodiments, the ring is a heterocycle. The term “heterocycle” refers to heteroaromatic and heteroalicyclic groups containing one to four heteroatoms each selected from O, S and N, wherein each heterocyclic group has from 4 to 10 atoms in its ring system, and with the proviso that the ring of said group does not contain two adjacent 0 or S atoms. Non-aromatic heterocyclic groups include groups having only 3 atoms in their ring system, but aromatic heterocyclic groups must have at least 5 atoms in their ring system. The heterocyclic groups include benzo-fused ring systems. An example of a 3-membered heterocyclic group is aziridinyl. An example of a 4-membered heterocyclic group is azetidinyl (derived from azetidine). An example of a 5-membered heterocyclic group is thiazolyl. An example of a 6-membered heterocyclic group is pyridyl, and an example of a 10-membered heterocyclic group is quinolinyl. Examples of non-aromatic heterocyclic groups are pyrrolidinyl, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothienyl, tetrahydropyranyl, dihydropyranyl, tetrahydrothiopyranyl, piperidino, morpholino, thiomorpholino, thioxanyl, piperazinyl, azetidinyl, oxetanyl, thietanyl, homopiperidinyl, oxepanyl, thiepanyl, oxazepinyl, diazepinyl, thiazepinyl, 1,2,3,6-tetrahydropyridinyl, 2-pyrrolinyl, 3-pyrrolinyl, indolinyl, 2H-pyranyl, 4H-pyranyl, dioxanyl, 1,3-dioxolanyl, pyrazolinyl, dithianyl, dithiolanyl, dihydropyranyl, dihydrothienyl, dihydrofuranyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, 3-azabicyclo[3.1.0]hexanyl, 3-azabicyclo[4.1.0]heptanyl, 3H-indolyl and quinolizinyl. Examples of aromatic heterocyclic groups are pyridinyl, imidazolyl, pyrimidinyl, pyrazolyl, triazolyl, pyrazinyl, tetrazolyl, furyl, thienyl, isoxazolyl, thiazolyl, oxazolyl, isothiazolyl, pyrrolyl, quinolinyl, isoquinolinyl, indolyl, benzimidazolyl, benzofuranyl, cinnolinyl, indazolyl, indolizinyl, phthalazinyl, pyridazinyl, triazinyl, isoindolyl, pteridinyl, purinyl, oxadiazolyl, thiadiazolyl, furazanyl, benzofurazanyl, benzothiophenyl, benzothiazolyl, benzoxazolyl, quinazolinyl, quinoxalinyl, naphthyridinyl, and furopyridinyl. The foregoing groups, may be C-attached or N-attached where such is possible. For instance, a group derived from pyrrole may be pyrrol-1-yl (N-attached) or pyrrol-3-yl (C-attached). Further, a group derived from imidazole may be imidazol-1-yl or imidazol-3-yl (both N-attached) or imidazol-2-yl, imidazol-4-yl or imidazol-5-yl (all C-attached). The heterocyclic groups include benzo-fused ring systems and ring systems substituted with one or two oxo (═O) moieties such as pyrrolidin-2-one. Depending on the structure, a heterocycle group can be a monoradical or a diradical (i.e., a heterocyclene group).

In some embodiments, the ring is fused. The term “fused” refers to structures in which two or more rings share one or more bonds. In some embodiments, the ring is a dimer. In some embodiments, the ring is a trimer. In some embodiments, the ring is a substituted.

The term “carbocyclic” or “carbocycle” refers to a ring wherein each of the atoms forming the ring is a carbon atom. Carbocycle includes aryl and cycloalkyl. The term thus distinguishes carbocycle from heterocycle (“heterocyclic”) in which the ring backbone contains at least one atom which is different from carbon (i.e., a heteroatom). Heterocycle includes heteroaryl and heterocycloalkyl. Carbocycles and heterocycles can be optionally substituted.

In some embodiments, the linker is substituted. The term “optionally substituted” or “substituted” means that the referenced group may be substituted with one or more additional group(s) individually and independently selected from C₁-C₆alkyl, C₃-C₈Cycloalkyl, aryl, heteroaryl, C₂-C₆heteroalicyclic, hydroxy, C₁-C₆alkoxy, aryloxy, C₁-C₆alkylthio, arylthio, C₁-C₆alkylsulfoxide, arylsulfoxide, C₁-C₆alkylsulfone, arylsulfone, cyano, halo, C₂-C₈acyl, C₂-C₈acyloxy, nitro, C₁-C₆haloalkyl, C₁-C₆fluoroalkyl, and amino, including C₁-C₆alkylamino, and the protected derivatives thereof. By way of example, an optional substituents may be LsRs, wherein each Ls is independently selected from a bond, —O-, —C(═O)—, —S—, —S(═O)—, —S(═O)₂—, —NH—, —NHC(═O)—, —C(═O)NH—, S(═O)₂NH-, —NHS(═O)2-, —OC(═O)NH—, —NHC(═O)O-, —(C₁-C₆alkyl)-, or —(C₂-C₆alkenyl)-; and each Rs is independently selected from H, (C₁-C₄alkyl), (C₃-C₈cycloalkyl), heteroaryl, aryl, and C₁-C₆heteroalkyl. Optionally substituted non-aromatic groups may be substituted with one or more oxo (═O). The protecting groups that may form the protective derivatives of the above substituents are known to those of skill in the art.

In some embodiments, a selective delivery molecules disclosed herein comprises a single of linker. Use of a single mechanism to mediate uptake of both imaging and therapeutic cargoes is particularly valuable, because imaging with noninjurious tracer quantities can be used to test whether a subsequent therapeutic dose is likely to concentrate correctly in the target tissue.

In some embodiments, a selective delivery molecules disclosed herein comprises a plurality of linkers. Where a selective delivery molecule disclosed herein includes multiple X linkages, separation of portion A from the other portions of the molecule requires cleavage of all X linkages. Cleavage of multiple X linkers may be simultaneous or sequential. Multiple X linkages may include X linkages having different specificities, so that separation of portion A from the other portions of the molecule requires that more than one condition or environment (“extracellular signals”) be encountered by the molecule. Cleavage of multiple X X linkers thus serves as a detector of combinations of such extracellular signals. For example, a selective delivery molecule may include two linker portions Xa and Xb connecting basic portion B with acidic portion A. Both X linkers and Xb must be cleaved before acidic portion A is separated from basic portion B allowing entry of portion B and cargo moiety C (if any) to enter a cell. It will be understood that a linker region may link to either a basic portion B or a cargo moiety C independently of another linker that may be present, and that, where desired, more than two linker regions X may be included.

Combinations of two or more X linkers may be used to further modulate the targeting and delivery of molecules to desired cells, tissue or regions. Combinations of extracellular signals are used to widen or narrow the specificity of the cleavage of X linkers if desired. Where multiple X linkers are linked in parallel, the specificity of cleavage is narrowed, since each X linker must be cleaved before portion A may separate from the remainder of the molecule. Where multiple X linkers are linked in series, the specificity of cleavage is broadened, since cleavage on any one X linker allows separation of portion A from the remainder of the molecule. For example, in order to detect either a protease OR hypoxia (i.e., to cleave X in the presence of either protease or hypoxia), a X linker is designed to place the protease-sensitive and reduction-sensitive sites in tandem, so that cleavage of either would suffice to allow separation of the acidic portion A. Alternatively, in order to detect the presence of both a protease AND hypoxia (i.e., to cleave X in the presence of both protease and hypoxia but not in the presence of only one alone), a X linker is designed to place the protease sensitive site between at least one pair of cysteines that are disulfide-bonded to each other. In that case, both protease cleavage and disulfide reduction are required in order to allow separation of portion A.

Exemplary Selective Delivery Molecules

Disclosed herein, in certain embodiments, are selective delivery molecules according to SDM-14.

Disclosed herein, in certain embodiments, are selective delivery molecules according to SDM-15.

Disclosed herein, in certain embodiments, are selective delivery molecules according to SDM-23.

Disclosed herein, in certain embodiments, are selective delivery molecules according to SDM-24.

Disclosed herein, in certain embodiments, are selective delivery molecules according to SDM-25.

Disclosed herein, in certain embodiments, are selective delivery molecules according to SDM-26.

Disclosed herein, in certain embodiments, are selective delivery molecules according to SDM-27.

Disclosed herein, in certain embodiments, are selective delivery molecules according to SDM-32.

Disclosed herein, in certain embodiments, are selective delivery molecules according to SDM-35.

Disclosed herein, in certain embodiments, are peptides according to Peptide P-3.

Disclosed herein, in certain embodiments, are selective delivery molecules according to SDM-14.

Disclosed herein, in certain embodiments, are selective delivery molecules according to SDM-15.

Disclosed herein, in certain embodiments, are selective delivery molecules according to SDM-23.

Disclosed herein, in certain embodiments, are selective delivery molecules according to SDM-24.

Disclosed herein, in certain embodiments, are selective delivery molecules according to SDM-25.

Disclosed herein, in certain embodiments, are selective delivery molecules according to SDM-26.

Disclosed herein, in certain embodiments, are selective delivery molecules according to SDM-27.

Disclosed herein, in certain embodiments, are selective delivery molecules according to SDM-32.

Disclosed herein, in certain embodiments, are selective delivery molecules according to SDM-35.

In some embodiments, the selective delivery molecule has a structure selected from: SDM-1, SDM-2, SDM-3, SDM-4, SDM-5, SDM-6, SDM-7, SDM-8, SDM-9, SDM-10, SDM-11, SDM-12, SDM-13, SDM-14, SDM-15, SDM-16, SDM-17, SDM-18, SDM-19, SDM-20, SDM-21, SDM-22, SDM-23, SDM-24, SDM-25, SDM-26, SDM-27, SDM-28, SDM-29, SDM-30, SDM-31, SDM-32, SDM-33, SDM-34, SDM-35, SDM-36, SDM-37, SDM-38, SDM-39, and SDM-40.

FURTHER MODIFICATIONS

In some embodiments, the targeting molecules of the present invention are optionally conjugated to high molecular weight molecules that increase the multivalency and avidity of labeling. In some embodiments, the high molecular weight molecules are water-soluble polymers. Examples of suitable water-soluble polymers include, but are not limited to, peptides, saccharides, poly(vinyls), poly(ethers), poly(amines), poly(carboxylic acids) and the like. In some embodiments, the water-soluble polymer is dextran, polyethylene glycol (PEG), polyoxyalkylene, polysialic acid, starch, or hydroxyethyl starch. Any suitable method is used to conjugate peptides to water-soluble polymers (see Hermanson G., Bioconjugate Techniques 2^(nd) Ed., Academic Press, Inc. 2008).

Pharmaceutical Compositions

Disclosed herein, in certain embodiments, are pharmaceutical compositions comprising a selective delivery molecule of Formula I, having the structure: [D_(A)-c _(A)]-A-[c _(M)-M]-X-B-[c _(B)-D_(B)]  Formula I wherein,

-   -   X is a cleavable linker;     -   A is a peptide with a sequence comprising 5 to 9 acidic amino         acids;     -   B is a peptide with a sequence comprising 7 to 9 basic amino         acids;     -   c_(A), c_(B), and c_(M) each independently comprise 0-1 amino         acid;     -   M is a macromolecule; and     -   D_(A) and D_(B) are each independently selected from an imaging         agent and a therapeutic; and         wherein [c_(M)-M] is bound to at any position on A or X,         [D_(A)-c_(A)] is bound to any amino acid on A, and [c_(B)-D_(B)]         is bound to any amino acid on B.         In some embodiments, A and B do not have an equal number of         acidic and basic amino acids. In some embodiments, the number of         basic amino acids in B is greater than the number of acidic         amino acids in A. In some embodiments, A is a peptide comprising         5 or 9 consecutive glutamates (SEQ ID NO: 3). In some         embodiments, B is a peptide comprising 8 or 9 consecutive         arginines (SEQ ID NO: 4). In some embodiments, A is a peptide         comprising 5 or 9 consecutive glutamates (SEQ ID NO: 3) and B is         a peptide comprising 8 or 9 consecutive arginines (SEQ ID NO:         4). In some embodiments, A is a peptide comprising 5 consecutive         glutamates (SEQ ID NO: 5) and B is a peptide comprising 8         consecutive arginines (SEQ ID NO: 6). In some embodiments,         c_(A), c_(B), and c_(M) are each independently a 0-1 amino acid.         In some embodiments, c_(A), c_(B), and c_(M) are each         independently selected from a naturally-occurring amino acid or         a non-naturally-occurring amino acid. In some embodiments,         c_(A), c_(B), and c_(M) are each independently selected from a D         amino acid, a L amino acid, an α-amino acid, a β-amino acid, or         a γ-amino acid. In some embodiments, c_(A), c_(B), and c_(M) are         each independently selected from any amino acid having a free         thiol group, any amino acid having a N-terminal amine group, and         any amino acid with a side chain capable of forming an oxime or         hydrazone bond upon reaction with a hydroxylamine or hydrazine         group. In some embodiments, c_(A), c_(B), and c_(M) are each         independently selected from D-cysteine, D-glutamate, lysine, and         para-4-acetyl L-phenylalanine. In some embodiments, c_(B) is any         amino acid having a free thiol group. In some embodiments, c_(B)         is D-cysteine. In some embodiments, c_(A) is any amino acid         having a N-terminal amine group. In some embodiments, c_(A) is         D-glutamate. In some embodiments, c_(A) is lysine. In some         embodiments, c_(M) is any amino acid with a side chain capable         of forming an oxime or hydrazone bond upon reaction with a         hydroxylamine or hydrazine group. In some embodiments, c_(M) is         para-4-acetyl L-phenylalanine. In some embodiments, X is         cleavable by a protease. In some embodiments, X is cleavable by         a matrix metalloproteinase. In some embodiments, X comprises an         amino acid sequence that is cleavable by MMP2, MMP7, MMP9, or         MMP14. In some embodiments, X comprises a peptide linkage. In         some embodiments, X comprises an amino acid sequence selected         from: PLGLAG (SEQ ID NO: 2), PLG-C(me)-AG (SEQ ID NO: 1),         RPLALWRS (SEQ ID NO: 7), ESPAYYTA (SEQ ID NO: 8), DPRSFL (SEQ ID         NO: 9), PPRSFL (SEQ ID NO: 10), RLQLKL (SEQ ID NO: 11), and         RLQLK(Ac) (SEQ ID NO: 12). In some embodiments, X comprises the         amino acid sequence PLGLAG (SEQ ID NO: 2). In some embodiments,         X comprises the amino acid sequence PLG-C(me)-AG (SEQ ID NO: 1).         In some embodiments, X comprises the amino acid sequence         RPLALWRS (SEQ ID NO: 7). In some embodiments, X comprises the         amino acid sequence DPRSFL (SEQ ID NO: 9). In some embodiments,         X comprises the amino acid sequence RLQLKL (SEQ ID NO: 11). In         some embodiments, X comprises the amino acid sequence RLQLK(Ac)         (SEQ ID NO: 12). In some embodiments, M is selected from a         protein, a natural polymer, a synthetic polymer, or a dendrimer.         In some embodiments, M is selected from dextran, a PEG polymer,         albumin, or a combination thereof. In some embodiments, M is a         PEG. In some embodiments, M is selected from PEG 5 kDa, PEG 12         kDa, PEG 20 kDa, PEG 30 kDa, and PEG40 kDa. In some embodiments,         D_(A) and D_(B) are a pair of acceptor and donor fluorescent         moieties that are capable of undergoing Försters/fluorescence         resonance energy transfer with the other. In some embodiments,         D_(A) and D_(B) are Cy5 and Cy7. In some embodiments, D_(A) and         D_(B) are Cy5 and IRDye750. In some embodiments, D_(A) and D_(B)         are Cy5 and IRDye800. In some embodiments, D_(A) and D_(B) are         Cy5 and ICG. In some embodiments, D_(A) and D_(B) are a         fluorescent moiety and a fluorescence-quenching moiety. In some         embodiments, the molecule of Formula I is: SDM-14, SDM-15,         SDM-23, SDM-24, SDM-25, SDM-26, SDM-27, SDM-32, or SDM-35.

Disclosed herein, in certain embodiments, are pharmaceutical compositions comprising a selective delivery molecule of Formula I, having the structure: [D_(A)-c _(A)]-A-[c _(M)-M]-X-B-[c _(B)-D_(B)]  Formula I wherein,

-   -   X is a cleavable linker;     -   A is a peptide with a sequence comprising 5 to 9 acidic amino         acids;     -   B is a peptide with a sequence comprising 7 to 9 basic amino         acids;     -   c_(A), c_(B), and c_(M) each independently comprise 0-1 amino         acid;     -   M is a polyethylene glycol (PEG) polymer; and     -   D_(A) and D_(B) are each independently an imaging agent; and     -   wherein [c_(M)-M] is bound to at any position on A or X,         [D_(A)-c_(A)] is bound to any amino acid on A, and [c_(B)-D_(B)]         is bound to any amino acid on B.         In some embodiments, A and B do not have an equal number of         acidic and basic amino acids. In some embodiments, the number of         basic amino acids in B is greater than the number of acidic         amino acids in A. In some embodiments, A is a peptide comprising         5 or 9 consecutive glutamates (SEQ ID NO: 3). In some         embodiments, B is a peptide comprising 8 or 9 consecutive         arginines (SEQ ID NO: 4). In some embodiments, A is a peptide         comprising 5 or 9 consecutive glutamates (SEQ ID NO: 3) and B is         a peptide comprising 8 or 9 consecutive arginines (SEQ ID NO:         4). In some embodiments, A is a peptide comprising 5 consecutive         glutamates (SEQ ID NO: 5) and B is a peptide comprising 8         consecutive arginines (SEQ ID NO: 6). In some embodiments,         c_(A), c_(B), and c_(M) are each independently a 0-1 amino acid.         In some embodiments, c_(A), c_(B), and c_(M) are each         independently selected from a naturally-occurring amino acid or         a non-naturally-occurring amino acid. In some embodiments,         c_(A), c_(B), and c_(M) are each independently selected from a D         amino acid, a L amino acid, an α-amino acid, a β-amino acid, or         a γ-amino acid. In some embodiments, c_(A), c_(B), and c_(M) are         each independently selected from any amino acid having a free         thiol group, any amino acid having a N-terminal amine group, and         any amino acid with a side chain capable of forming an oxime or         hydrazone bond upon reaction with a hydroxylamine or hydrazine         group. In some embodiments, c_(A), c_(B), and c_(M) are each         independently selected from D-cysteine, D-glutamate, lysine, and         para-4-acetyl L-phenylalanine. In some embodiments, c_(B) is any         amino acid having a free thiol group. In some embodiments, c_(B)         is D-cysteine. In some embodiments, c_(A) is any amino acid         having a N-terminal amine group. In some embodiments, c_(A) is         D-glutamate. In some embodiments, c_(A) is lysine. In some         embodiments, c_(M) is any amino acid with a side chain capable         of forming an oxime or hydrazone bond upon reaction with a         hydroxylamine or hydrazine group. In some embodiments, c_(M) is         para-4-acetyl L-phenylalanine. In some embodiments, X is         cleavable by a protease. In some embodiments, X is cleavable by         a matrix metalloproteinase. In some embodiments, X comprises an         amino acid sequence that is cleavable by MMP2, MMP7, MMP9, or         MMP14. In some embodiments, X comprises a peptide linkage. In         some embodiments, X comprises an amino acid sequence selected         from: PLGLAG (SEQ ID NO: 2), PLG-C(me)-AG (SEQ ID NO: 1),         RPLALWRS (SEQ ID NO: 7), ESPAYYTA (SEQ ID NO: 8), DPRSFL (SEQ ID         NO: 9), PPRSFL (SEQ ID NO: 10), RLQLKL (SEQ ID NO: 11), and         RLQLK(Ac) (SEQ ID NO: 12). In some embodiments, X comprises the         amino acid sequence PLGLAG (SEQ ID NO: 2). In some embodiments,         X comprises the amino acid sequence PLG-C(me)-AG (SEQ ID NO: 1).         In some embodiments, X comprises the amino acid sequence         RPLALWRS (SEQ ID NO: 7). In some embodiments, X comprises the         amino acid sequence DPRSFL (SEQ ID NO: 9). In some embodiments,         X comprises the amino acid sequence PPRSFL (SEQ ID NO: 10). In         some embodiments, X comprises the amino acid sequence RLQLKL         (SEQ ID NO: 11). In some embodiments, X comprises the amino acid         sequence RLQLK(Ac) (SEQ ID NO: 12). In some embodiments, D_(A)         and D_(B) are a pair of acceptor and donor fluorescent moieties         that are capable of undergoing Försters/fluorescence resonance         energy transfer with the other. In some embodiments, D_(A) and         D_(B) are Cy5 and Cy7. In some embodiments, D_(A) and D_(B) are         Cy5 and IRDye750. In some embodiments, D_(A) and D_(B) are Cy5         and IRDye800. In some embodiments, D_(A) and D_(B) are Cy5 and         ICG. In some embodiments, D_(A) and D_(B) are a fluorescent         moiety and a fluorescence-quenching moiety. In some embodiments,         the molecule of Formula I is: SDM-14, SDM-15, SDM-23, SDM-24,         SDM-25, SDM-26, SDM-27, SDM-32; or SDM-35.

Disclosed herein, in certain embodiments, are pharmaceutical compositions comprising a selective delivery molecule of Formula I, having the structure: [D_(A)-c _(A)]-A-[c _(M)-M]-X-B-[c _(B)-D_(B)]  Formula I wherein,

-   -   X is a peptide linker cleavable by a matrix metalloproteinase;     -   A is a peptide with a sequence comprising 5 or 9 consecutive         glutamates (SEQ ID NO: 3);     -   B is a peptide with a sequence comprising 8 or 9 consecutive         arginines (SEQ ID NO: 4);     -   c_(A), c_(B), and c_(M) each independently comprise 0-1 amino         acid;     -   M is a polyethylene glycol (PEG) polymer; and     -   D_(A) and D_(B) are a pair of acceptor and donor fluorescent         moieties that are capable of undergoing Försters/fluorescence         resonance energy transfer with the other; and         wherein [c_(M)-M] is bound to at any position on A or X,         [D_(A)-c_(A)] is bound to any amino acid on A, and [c_(B)-D_(B)]         is bound to any amino acid on B.         In some embodiments, A and B do not have an equal number of         acidic and basic amino acids. In some embodiments, c_(A), c_(B),         and c_(M) are each independently selected from any amino acid         having a free thiol group, any amino acid having a N-terminal         amine group, and any amino acid with a side chain capable of         forming an oxime or hydrazone bond upon reaction with a         hydroxylamine or hydrazine group. In some embodiments, c_(A),         c_(B), and c_(M) are each independently a 0-1 amino acid. In         some embodiments, c_(A), c_(B), and c_(M) are each independently         selected from D-cysteine, D-glutamate, lysine, and para-4-acetyl         L-phenylalanine. In some embodiments, c_(B) is any amino acid         having a free thiol group. In some embodiments, c_(B) is         D-cysteine. In some embodiments, c_(A) is any amino acid having         a N-terminal amine group. In some embodiments, c_(A) is         D-glutamate. In some embodiments, c_(M) is any amino acid with a         side chain capable of forming an oxime or hydrazone bond upon         reaction with a hydroxylamine or hydrazine group. In some         embodiments, c_(M) is para-4-acetyl L-phenylalanine. In some         embodiments, X comprises the amino acid sequence PLGLAG (SEQ ID         NO: 2). In some embodiments, X comprises the amino acid sequence         PLG-C(me)-AG (SEQ ID NO: 1). In some embodiments, D_(A) and         D_(B) are Cy5 and Cy7. In some embodiments, D_(A) and D_(B) are         Cy5 and Cy7.

Disclosed herein, in certain embodiments, are pharmaceutical compositions comprising a selective delivery molecule of Formula I, having the structure: [D_(A)-c _(A)]-A-[c _(M)-M]-X-B-[c _(B)-D_(B)]  Formula I wherein,

-   -   X is a peptide linker cleavable by a matrix metalloproteinase;     -   A is a peptide with a sequence comprising 5 consecutive         glutamates (SEQ ID NO: 5);     -   B is a peptide with a sequence comprising 8 consecutive         arginines (SEQ ID NO: 6);     -   c_(A), c_(B), and c_(M) each independently comprise 0-1 amino         acid;     -   M is a polyethylene glycol (PEG) polymer; and     -   D_(A) and D_(B) are a pair of acceptor and donor fluorescent         moieties that are capable of undergoing Försters/fluorescence         resonance energy transfer with the other; and         wherein [c_(M)-M] is bound to at any position on A or X,         [D_(A)-c_(A)] is bound to any amino acid on A, and [c_(B)-D_(B)]         is bound to any amino acid on B.         In some embodiments, c_(A), c_(B), and c_(M) are each         independently a 0-1 amino acid. In some embodiments, c_(A),         c_(B), and c_(M) are each independently selected from any amino         acid having a free thiol group, any amino acid having a         N-terminal amine group, and any amino acid with a side chain         capable of forming an oxime or hydrazone bond upon reaction with         a hydroxylamine or hydrazine group. In some embodiments, c_(A),         c_(B), and c_(M) are each independently selected from         D-cysteine, D-glutamate, lysine, and para-4-acetyl         L-phenylalanine. In some embodiments, c_(B) is any amino acid         having a free thiol group. In some embodiments, c_(B) is         D-cysteine. In some embodiments, c_(A) is any amino acid having         a N-terminal amine group. In some embodiments, c_(A) is         D-glutamate. In some embodiments, c_(M) is any amino acid with a         side chain capable of forming an oxime or hydrazone bond upon         reaction with a hydroxylamine or hydrazine group. In some         embodiments, c_(M) is para-4-acetyl L-phenylalanine. In some         embodiments, X comprises the amino acid sequence PLGLAG (SEQ ID         NO: 2). In some embodiments, X comprises the amino acid sequence         PLG-C(me)-AG (SEQ ID NO: 1). In some embodiments, X comprises         the amino acid sequence RPLALWRS (SEQ ID NO: 7). In some         embodiments, D_(A) and D_(B) are Cy5 and Cy7. In some         embodiments, D_(A) and D_(B) are Cy5 and IRDye750. In some         embodiments, D_(A) and D_(B) are Cy5 and IRDye800. In some         embodiments, D_(A) and D_(B) are Cy5 and ICG.

Disclosed herein, in certain embodiments, are pharmaceutical compositions comprising a selective delivery molecule of Formula I, having the structure: [D_(A)-c _(A)]-A-[c _(M)-M]-X-B-[c _(B)-D_(B)]  Formula I wherein,

-   -   X is a peptide linker cleavable by a matrix metalloproteinase;     -   A is a peptide with a sequence comprising 9 consecutive         glutamates (SEQ ID NO: 13);     -   B is a peptide with a sequence comprising 9 consecutive         arginines (SEQ ID NO: 14);     -   c_(A), c_(B), and c_(M) each independently comprise 0-1 amino         acid;     -   M is a polyethylene glycol (PEG) polymer; and     -   D_(A) and D_(B) are a pair of acceptor and donor fluorescent         moieties that are capable of undergoing Försters/fluorescence         resonance energy transfer with the other; and         wherein [c_(M)-M] is bound to at any position on A or X,         [D_(A)-c_(A)] is bound to any amino acid on A, and [c_(B)-D_(B)]         is bound to any amino acid on B.         In some embodiments, c_(A), c_(B), and c_(M) are each         independently a 0-1 amino acid. In some embodiments, c_(A),         c_(B), and c_(M) are each independently selected from any amino         acid having a free thiol group, any amino acid having a         N-terminal amine group, and any amino acid with a side chain         capable of forming an oxime or hydrazone bond upon reaction with         a hydroxylamine or hydrazine group. In some embodiments, c_(A),         c_(B), and c_(M) are each independently selected from         D-cysteine, D-glutamate, lysine, and para-4-acetyl         L-phenylalanine. In some embodiments, c_(B) is any amino acid         having a free thiol group. In some embodiments, c_(B) is         D-cysteine. In some embodiments, c_(A) is any amino acid having         a N-terminal amine group. In some embodiments, c_(A) is         D-glutamate. In some embodiments, c_(M) is any amino acid with a         side chain capable of forming an oxime or hydrazone bond upon         reaction with a hydroxylamine or hydrazine group. In some         embodiments, c_(M) is para-4-acetyl L-phenylalanine. In some         embodiments, X comprises the amino acid sequence PLGLAG (SEQ ID         NO: 2). In some embodiments, X comprises the amino acid sequence         PLG-C(me)-AG (SEQ ID NO: 1). In some embodiments, X comprises         the amino acid sequence RPLALWRS (SEQ ID NO: 7). In some         embodiments, D_(A) and D_(B) are Cy5 and Cy7. In some         embodiments, D_(A) and D_(B) are Cy5 and IRDye750. In some         embodiments, D_(A) and D_(B) are Cy5 and IRDye800. In some         embodiments, D_(A) and D_(B) are Cy5 and ICG.

Disclosed herein, in certain embodiments, are pharmaceutical compositions comprising a selective delivery molecule according to SDM-14.

Disclosed herein, in certain embodiments, are pharmaceutical compositions comprising a selective delivery molecule according to SDM-15.

Disclosed herein, in certain embodiments, are pharmaceutical compositions comprising a selective delivery molecule according to SDM-23.

Disclosed herein, in certain embodiments, are pharmaceutical compositions comprising a selective delivery molecule according to SDM-24.

Disclosed herein, in certain embodiments, are pharmaceutical compositions comprising a selective delivery molecule according to SDM-25.

Disclosed herein, in certain embodiments, are pharmaceutical compositions comprising a selective delivery molecule according to SDM-26.

Disclosed herein, in certain embodiments, are pharmaceutical compositions comprising a selective delivery molecule according to SDM-27.

Disclosed herein, in certain embodiments, are pharmaceutical compositions comprising a selective delivery molecule according to SDM-32.

Disclosed herein, in certain embodiments, are pharmaceutical compositions comprising a selective delivery molecule according to SDM-35.

Disclosed herein, in certain embodiments, are selective delivery molecules according to SDM-14.

Disclosed herein, in certain embodiments, are selective delivery molecules according to SDM-15.

Disclosed herein, in certain embodiments, are selective delivery molecules according to SDM-23.

Disclosed herein, in certain embodiments, are selective delivery molecules according to SDM-24.

Disclosed herein, in certain embodiments, are selective delivery molecules according to SDM-25.

Disclosed herein, in certain embodiments, are selective delivery molecules according to SDM-26.

Disclosed herein, in certain embodiments, are selective delivery molecules according to SDM-27.

Disclosed herein, in certain embodiments, are selective delivery molecules according to SDM-32.

Disclosed herein, in certain embodiments, are selective delivery molecules according to SDM-35.

Pharmaceutical compositions herein are formulated using one or more physiologically acceptable carriers including excipients and auxiliaries which facilitate processing of the active agents into preparations which are used pharmaceutically. Proper formulation is dependent upon the route of administration chosen. A summary of pharmaceutical compositions is found, for example, in Remington: The Science and Practice of Pharmacy, Nineteenth Ed (Easton, Pa.: Mack Publishing Company, 1995); Hoover, John E., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa. 1975; Liberman, H. A. and Lachman, L., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, N.Y., 1980; and Pharmaceutical Dosage Forms and Drug Delivery Systems, Seventh Ed. (Lippincott Williams & Wilkins, 1999).

In certain embodiments, a pharmaceutical composition disclosed herein further comprises a pharmaceutically acceptable diluent(s), excipient(s), or carrier(s). In some embodiments, the pharmaceutical compositions includes other medicinal or pharmaceutical agents, carriers, adjuvants, such as preserving, stabilizing, wetting or emulsifying agents, solution promoters, salts for regulating the osmotic pressure, and/or buffers. In addition, the pharmaceutical compositions also contain other therapeutically valuable substances.

In certain embodiments, a pharmaceutical composition disclosed herein is administered to a subject by any suitable administration route, including but not limited to, parenteral (intravenous, subcutaneous, intraperitoneal, intramuscular, intravascular, intrathecal, intravitreal, infusion, or local) administration.

Formulations suitable for intramuscular, subcutaneous, peritumoral, or intravenous injection include physiologically acceptable sterile aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Examples of suitable aqueous and non-aqueous carriers, diluents, solvents, or vehicles including water, ethanol, polyols (propyleneglycol, polyethylene-glycol, glycerol, cremophor and the like), suitable mixtures thereof, vegetable oils (such as olive oil) and injectable organic esters such as ethyl oleate. Proper fluidity is maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Formulations suitable for subcutaneous injection also contain optional additives such as preserving, wetting, emulsifying, and dispensing agents.

For intravenous injections, an active agent is optionally formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological saline buffer.

Parenteral injections optionally involve bolus injection or continuous infusion. Formulations for injection are optionally presented in unit dosage form, e.g., in ampoules or in multi dose containers, with an added preservative. In some embodiments, the pharmaceutical composition described herein are in a form suitable for parenteral injection as a sterile suspensions, solutions or emulsions in oily or aqueous vehicles, and contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Pharmaceutical formulations for parenteral administration include aqueous solutions of an active agent in water soluble form. Additionally, suspensions are optionally prepared as appropriate oily injection suspensions.

In some embodiments, the pharmaceutical composition described herein is in unit dosage forms suitable for single administration of precise dosages. In unit dosage form, the formulation is divided into unit doses containing appropriate quantities of an active agent disclosed herein. In some embodiments, the unit dosage is in the form of a package containing discrete quantities of the formulation. Non-limiting examples are packaged tablets or capsules, and powders in vials or ampoules. In some embodiments, aqueous suspension compositions are packaged in single-dose non-reclosable containers. Alternatively, multiple-dose reclosable containers are used, in which case it is typical to include a preservative in the composition. By way of example only, formulations for parenteral injection are presented in unit dosage form, which include, but are not limited to ampoules, or in multi dose containers, with an added preservative.

Methods of Use

The selective delivery molecules of Formula I allow the targeted delivery of therapeutic agents and/or imaging agents to specific cells and/or tissues. The molecules comprise a basic peptide sequence (B) which is designed to be transported across a cellular membrane, an acidic peptide sequence (A) which inhibits uptake of peptide B into cells, a linker X which is cleavable under specific conditions, cargo moieties (at least D_(A) and D_(B)) bound to peptides A and B, or X and a macromolecular carrier. In some embodiments, cleavage of the linker X linker frees peptide B from peptide A and allows the transport of peptide B (and any cargo attached thereto) across a cellular membrane. In some embodiments, the selective delivery molecules of Formula I enable targeted delivery of one or more cargos (e.g., therapeutic agents or imaging agents) to a cell tissue.

Disclosed herein, in certain embodiments, are methods of delivering cargo to a tissue of interest, comprising contacting the tissue of interest with a molecule of Formula I: [D_(A)-c _(A)]-A-[c _(M)-M]-X-B-[c _(B)-D_(B)]  Formula I wherein,

-   -   X is a cleavable linker;     -   A is a peptide with a sequence comprising 5 to 9 acidic amino         acids;     -   B is a peptide with a sequence comprising 7 to 9 basic amino         acids;     -   c_(A), c_(B), and c_(M) each independently comprise 0-1 amino         acid;     -   M is a macromolecule; and     -   D_(A) and D_(B) are each independently selected from an imaging         agent and a therapeutic; and         wherein [c_(M)-M] is bound to at any position on A or X,         [D_(A)-c_(A)] is bound to any amino acid on A, and [c_(B)-D_(B)]         is bound to any amino acid on B.         In some embodiments, A and B do not have an equal number of         acidic and basic amino acids. In some embodiments, the number of         basic amino acids in B is greater than the number of acidic         amino acids in A. In some embodiments, A is a peptide comprising         5 or 9 consecutive glutamates (SEQ ID NO: 3). In some         embodiments, B is a peptide comprising 8 or 9 consecutive         arginines (SEQ ID NO: 4). In some embodiments, A is a peptide         comprising 5 or 9 consecutive glutamates (SEQ ID NO: 3) and B is         a peptide comprising 8 or 9 consecutive arginines (SEQ ID NO:         4). In some embodiments, A is a peptide comprising 5 consecutive         glutamates (SEQ ID NO: 5) and B is a peptide comprising 8         consecutive arginines (SEQ ID NO: 6). In some embodiments,         c_(A), c_(B), and c_(M) are each independently a 0-1 amino acid.         In some embodiments, c_(A), c_(B), and c_(M) are each         independently selected from a naturally-occurring amino acid or         a non-naturally-occurring amino acid. In some embodiments,         c_(A), c_(B), and c_(M) are each independently selected from a D         amino acid, a L amino acid, an α-amino acid, a β-amino acid, or         a γ-amino acid. In some embodiments, c_(A), c_(B), and c_(M) are         each independently selected from any amino acid having a free         thiol group, any amino acid having a N-terminal amine group, and         any amino acid with a side chain capable of forming an oxime or         hydrazone bond upon reaction with a hydroxylamine or hydrazine         group. In some embodiments, c_(A), c_(B), and c_(M) are each         independently selected from D-cysteine, D-glutamate, lysine, and         para-4-acetyl L-phenylalanine. In some embodiments, c_(B) is any         amino acid having a free thiol group. In some embodiments, c_(B)         is D-cysteine. In some embodiments, c_(A) is any amino acid         having a N-terminal amine group. In some embodiments, c_(A) is         D-glutamate. In some embodiments, c_(A) is lysine. In some         embodiments, c_(M) is any amino acid with a side chain capable         of forming an oxime or hydrazone bond upon reaction with a         hydroxylamine or hydrazine group. In some embodiments, c_(M) is         para-4-acetyl L-phenylalanine. In some embodiments, X is         cleavable by a protease. In some embodiments, X is cleavable by         a matrix metalloproteinase. In some embodiments, X comprises an         amino acid sequence that is cleavable by MMP2, MMP7, MMP9, or         MMP14. In some embodiments, X comprises a peptide linkage. In         some embodiments, X comprises an amino acid sequence selected         from: PLGLAG (SEQ ID NO: 2), PLG-C(me)-AG (SEQ ID NO: 1),         RPLALWRS (SEQ ID NO: 7), ESPAYYTA (SEQ ID NO: 8), DPRSFL (SEQ ID         NO: 9), PPRSFL (SEQ ID NO: 10), RLQLKL (SEQ ID NO: 11), and         RLQLK(Ac) (SEQ ID NO: 12). In some embodiments, X comprises the         amino acid sequence PLGLAG (SEQ ID NO: 2). In some embodiments,         X comprises the amino acid sequence PLG-C(me)-AG (SEQ ID NO: 1).         In some embodiments, X comprises the amino acid sequence         RPLALWRS (SEQ ID NO: 7). In some embodiments, X comprises the         amino acid sequence DPRSFL (SEQ ID NO: 9). In some embodiments,         X comprises the amino acid sequence RLQLKL (SEQ ID NO: 11). In         some embodiments, X comprises the amino acid sequence RLQLK(Ac)         (SEQ ID NO: 12). In some embodiments, M is selected from a         protein, a natural polymer, a synthetic polymer, or a dendrimer.         In some embodiments, M is selected from dextran, a PEG polymer,         albumin, or a combination thereof. In some embodiments, M is a         PEG. In some embodiments, M is selected from PEG 5 kDa, PEG 12         kDa, PEG 20 kDa, PEG 30 kDa, and PEG40 kDa. In some embodiments,         D_(A) and D_(B) are a pair of acceptor and donor fluorescent         moieties that are capable of undergoing Försters/fluorescence         resonance energy transfer with the other. In some embodiments,         D_(A) and D_(B) are Cy5 and Cy7. In some embodiments, D_(A) and         D_(B) are Cy5 and IRDye750. In some embodiments, D_(A) and D_(B)         are Cy5 and IRDye800. In some embodiments, D_(A) and D_(B) are         Cy5 and ICG. In some embodiments, D_(A) and D_(B) are a         fluorescent moiety and a fluorescence-quenching moiety. In some         embodiments, the molecule of Formula I is: SDM-14, SDM-15,         SDM-23, SDM-24, SDM-25, SDM-26, SDM-27, SDM-32, or SDM-35.

Disclosed herein, in certain embodiments, are methods of delivering cargo to a tissue of interest, comprising contacting the tissue of interest with a molecule of Formula I: [D_(A)-c _(A)]-A-[c _(M)-M]-X-B-[c _(B)-D_(B)]  Formula I wherein,

-   -   X is a cleavable linker;     -   A is a peptide with a sequence comprising 5 to 9 acidic amino         acids;     -   B is a peptide with a sequence comprising 7 to 9 basic amino         acids;     -   c_(A), c_(B), and c_(M) each independently comprise 0-1 amino         acid;     -   M is a polyethylene glycol (PEG) polymer; and     -   D_(A) and D_(B) are each independently selected from imaging         agents and therapeutic agents; and     -   wherein [c_(M)-M] is bound to at any position on A or X,         [D_(A)-c_(A)] is bound to any amino acid on A, and [c_(B)-D_(B)]         is bound to any amino acid on B.         In some embodiments, A and B do not have an equal number of         acidic and basic amino acids. In some embodiments, the number of         basic amino acids in B is greater than the number of acidic         amino acids in A. In some embodiments, A is a peptide comprising         5 or 9 consecutive glutamates (SEQ ID NO: 3). In some         embodiments, B is a peptide comprising 8 or 9 consecutive         arginines (SEQ ID NO: 4). In some embodiments, A is a peptide         comprising 5 or 9 consecutive glutamates (SEQ ID NO: 3) and B is         a peptide comprising 8 or 9 consecutive arginines (SEQ ID NO:         4). In some embodiments, A is a peptide comprising 5 consecutive         glutamates (SEQ ID NO: 5) and B is a peptide comprising 8         consecutive arginines (SEQ ID NO: 6). In some embodiments,         c_(A), c_(B), and c_(M) are each independently a 0-1 amino acid.         In some embodiments, c_(A), c_(B), and c_(M) are each         independently selected from a naturally-occurring amino acid or         a non-naturally-occurring amino acid. In some embodiments,         c_(A), c_(B), and c_(M) are each independently selected from a D         amino acid, a L amino acid, an α-amino acid, a β-amino acid, or         a γ-amino acid. In some embodiments, c_(A), c_(B), and c_(M) are         each independently selected from any amino acid having a free         thiol group, any amino acid having a N-terminal amine group, and         any amino acid with a side chain capable of forming an oxime or         hydrazone bond upon reaction with a hydroxylamine or hydrazine         group. In some embodiments, c_(A), c_(B), and c_(M) are each         independently selected from D-cysteine, D-glutamate, lysine, and         para-4-acetyl L-phenylalanine. In some embodiments, c_(B) is any         amino acid having a free thiol group. In some embodiments, c_(B)         is D-cysteine. In some embodiments, c_(A) is any amino acid         having a N-terminal amine group. In some embodiments, c_(A) is         D-glutamate. In some embodiments, c_(A) is lysine. In some         embodiments, c_(M) is any amino acid with a side chain capable         of forming an oxime or hydrazone bond upon reaction with a         hydroxylamine or hydrazine group. In some embodiments, c_(M) is         para-4-acetyl L-phenylalanine. In some embodiments, X is         cleavable by a protease. In some embodiments, X is cleavable by         a matrix metalloproteinase. In some embodiments, X comprises an         amino acid sequence that is cleavable by MMP2, MMP7, MMP9, or         MMP14. In some embodiments, X comprises a peptide linkage. In         some embodiments, X comprises an amino acid sequence selected         from: PLGLAG (SEQ ID NO: 2), PLG-C(me)-AG (SEQ ID NO: 1),         RPLALWRS (SEQ ID NO: 7), ESPAYYTA (SEQ ID NO: 8), DPRSFL (SEQ ID         NO: 9), PPRSFL (SEQ ID NO: 10), RLQLKL (SEQ ID NO: 11), and         RLQLK(Ac) (SEQ ID NO: 12). In some embodiments, X comprises the         amino acid sequence PLGLAG (SEQ ID NO: 2). In some embodiments,         X comprises the amino acid sequence PLG-C(me)-AG (SEQ ID NO: 1).         In some embodiments, X comprises the amino acid sequence         RPLALWRS (SEQ ID NO: 7). In some embodiments, X comprises the         amino acid sequence DPRSFL (SEQ ID NO: 9). In some embodiments,         X comprises the amino acid sequence PPRSFL (SEQ ID NO: 10). In         some embodiments, X comprises the amino acid sequence RLQLKL         (SEQ ID NO: 11). In some embodiments, X comprises the amino acid         sequence RLQLK(Ac) (SEQ ID NO: 12).

Disclosed herein, in certain embodiments, are methods of delivering cargo to a tissue of interest, comprising contacting the tissue of interest with a molecule of Formula I: [D_(A)-c _(A)]-A-[c _(M)-M]-X-B-[c _(B)-D_(B)]  Formula I wherein,

-   -   X is a peptide linker cleavable by a matrix metalloproteinase;     -   A is a peptide with a sequence comprising 5 or 9 consecutive         glutamates (SEQ ID NO: 3);     -   B is a peptide with a sequence comprising 8 or 9 consecutive         arginines (SEQ ID NO: 4);     -   c_(A), c_(B), and c_(M) each independently comprise 0-1 amino         acid;     -   M is a polyethylene glycol (PEG) polymer; and     -   D_(A) and D_(B) are independently selected from imagining agents         and therapeutic agents; and         wherein [c_(M)-M] is bound to at any position on A or X,         [D_(A)-c_(A)] is bound to any amino acid on A, and [c_(B)-D_(B)]         is bound to any amino acid on B.         In some embodiments, A and B do not have an equal number of         acidic and basic amino acids. In some embodiments, c_(A), c_(B),         and c_(M) are each independently a 0-1 amino acid. In some         embodiments, c_(A), c_(B), and c_(M) are each independently         selected from any amino acid having a free thiol group, any         amino acid having a N-terminal amine group, and any amino acid         with a side chain capable of forming an oxime or hydrazone bond         upon reaction with a hydroxylamine or hydrazine group. In some         embodiments, c_(A), c_(B), and c_(M) are each independently         selected from D-cysteine, D-glutamate, lysine, and para-4-acetyl         L-phenylalanine. In some embodiments, c_(B) is any amino acid         having a free thiol group. In some embodiments, c_(B) is         D-cysteine. In some embodiments, c_(A) is any amino acid having         a N-terminal amine group. In some embodiments, c_(A) is         D-glutamate. In some embodiments, c_(M) is any amino acid with a         side chain capable of forming an oxime or hydrazone bond upon         reaction with a hydroxylamine or hydrazine group. In some         embodiments, c_(M) is para-4-acetyl L-phenylalanine. In some         embodiments, X comprises the amino acid sequence PLGLAG (SEQ ID         NO: 2). In some embodiments, X comprises the amino acid sequence         PLG-C(me)-AG (SEQ ID NO: 1).

Disclosed herein, in certain embodiments, are methods of delivering cargo to a tissue of interest, comprising contacting the tissue of interest with a molecule of Formula I: [D_(A)-c _(A)]-A-[c _(M)-M]-X-B-[c _(B)-D_(B)]  Formula I wherein,

-   -   X is a peptide linker cleavable by a matrix metalloproteinase;     -   A is a peptide with a sequence comprising 5 consecutive         glutamates (SEQ ID NO: 5);     -   B is a peptide with a sequence comprising 8 consecutive         arginines (SEQ ID NO: 6);     -   c_(A), c_(B), and c_(M) each independently comprise 0-1 amino         acid;     -   M is a polyethylene glycol (PEG) polymer; and     -   D_(A) and D_(B) are independently selected from imaging agents         and therapeutic agents; and         wherein [c_(M)-M] is bound to at any position on A or X,         [D_(A)-c_(A)] is bound to any amino acid on A, and [c_(B)-D_(B)]         is bound to any amino acid on B.         In some embodiments, c_(A), c_(B), and c_(M) are each         independently a 0-1 amino acid. In some embodiments, c_(A),         c_(B), and c_(M) are each independently selected from any amino         acid having a free thiol group, any amino acid having a         N-terminal amine group, and any amino acid with a side chain         capable of forming an oxime or hydrazone bond upon reaction with         a hydroxylamine or hydrazine group. In some embodiments, c_(A),         c_(B), and c_(M) are each independently selected from         D-cysteine, D-glutamate, lysine, and para-4-acetyl         L-phenylalanine. In some embodiments, c_(B) is any amino acid         having a free thiol group. In some embodiments, c_(B) is         D-cysteine. In some embodiments, c_(A) is any amino acid having         a N-terminal amine group. In some embodiments, c_(A) is         D-glutamate. In some embodiments, c_(M) is any amino acid with a         side chain capable of forming an oxime or hydrazone bond upon         reaction with a hydroxylamine or hydrazine group. In some         embodiments, c_(M) is para-4-acetyl L-phenylalanine. In some         embodiments, X comprises the amino acid sequence PLGLAG (SEQ ID         NO: 2). In some embodiments, X comprises the amino acid sequence         PLG-C(me)-AG (SEQ ID NO: 1). In some embodiments, X comprises         the amino acid sequence RPLALWRS (SEQ ID NO: 7).

Disclosed herein, in certain embodiments, are methods of delivering cargo to a tissue of interest, comprising contacting the tissue of interest with a molecule of Formula I: [D_(A)-c _(A)]-A-[c _(M)-M]-X-B-[c _(B)-D_(B)]  Formula I wherein,

-   -   X is a peptide linker cleavable by a matrix metalloproteinase;     -   A is a peptide with a sequence comprising 9 consecutive         glutamates (SEQ ID NO: 13);     -   B is a peptide with a sequence comprising 9 consecutive         arginines (SEQ ID NO: 14);     -   c_(A), c_(B), and c_(M) each independently comprise 0-1 amino         acid;     -   M is a polyethylene glycol (PEG) polymer; and     -   D_(A) and D_(B) are independently selected from imaging agents         and therapeutic agents; and         wherein [c_(M)-M] is bound to at any position on A or X,         [D_(A)-c_(A)] is bound to any amino acid on A, and [c_(B)-D_(B)]         is bound to any amino acid on B.         In some embodiments, c_(A), c_(B), and c_(M) are each         independently a 0-1 amino acid. In some embodiments, c_(A),         c_(B), and c_(M) are each independently selected from any amino         acid having a free thiol group, any amino acid having a         N-terminal amine group, and any amino acid with a side chain         capable of forming an oxime or hydrazone bond upon reaction with         a hydroxylamine or hydrazine group. In some embodiments, c_(A),         c_(B), and c_(M) are each independently selected from         D-cysteine, D-glutamate, lysine, and para-4-acetyl         L-phenylalanine. In some embodiments, c_(B) is any amino acid         having a free thiol group. In some embodiments, c_(B) is         D-cysteine. In some embodiments, c_(A) is any amino acid having         a N-terminal amine group. In some embodiments, c_(A) is         D-glutamate. In some embodiments, c_(M) is any amino acid with a         side chain capable of forming an oxime or hydrazone bond upon         reaction with a hydroxylamine or hydrazine group. In some         embodiments, c_(M) is para-4-acetyl L-phenylalanine. In some         embodiments, X comprises the amino acid sequence PLGLAG (SEQ ID         NO: 2). In some embodiments, X comprises the amino acid sequence         PLG-C(me)-AG (SEQ ID NO: 1). In some embodiments, X comprises         the amino acid sequence RPLALWRS (SEQ ID NO: 7).         Tissue of Interest

In some embodiments, the tissue of interest is cancerous tissue (or, cancer). In some embodiments, the cancerous tissue is: breast cancer tissue, colon cancer tissue, squamous cell carcinoma tissue, prostate cancer tissue, melanoma tissue, or thyroid cancer tissue. In some embodiments, the cancerous tissue is breast cancer tissue. In some embodiments, the cancerous tissue is colon cancer tissue.

In some embodiments, the cancer is AIDS-related cancers (e.g., AIDS-related lymphoma), anal cancer, basal cell carcinoma, bile duct cancer (e.g., extrahepatic), bladder cancer, bone cancer, (osteosarcoma and malignant fibrous histiocytoma), breast cancer, cervical cancer, colon cancer, colorectal cancer, endometrial cancer (e.g., uterine cancer), ependymoma, esophageal cancer, eye cancer (e.g., intraocular melanoma and retinoblastoma), gastric (stomach) cancer, germ cell tumor, (e.g., extracranial, extragonadal, ovarian), head and neck cancer, leukemia, lip and oral cavity cancer, liver cancer, lung cancer (e.g., small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung), ovarian cancer, pancreatic cancer, pituitary tumor, prostate cancer, renal cancer, skin cancer, small intestine cancer, squamous cell cancer, testicular cancer, throat cancer, thyroid cancer, urethral cancer, and post-transplant lymphoproliferative disorder (PTLD).

In some embodiments, the cancer is a lymphoid cancer (e.g., lymphoma).

In some embodiments, the cancer is a B-cell cancer. In some embodiments, the cancer is precursor B-cell cancers (e.g., precursor B-lymphoblastic leukemia/lymphoma) and peripheral B-cell cancers (e.g., B-cell chronic lymphocytic leukemia/prolymphocytic leukemia/small lymphocytic lymphoma (small lymphocytic (SL) NHL), lymphoplasmacytoid lymphoma/immunocytoma, mantel cell lymphoma, follicle center lymphoma, follicular lymphoma (e.g., cytologic grades: I (small cell), II (mixed small and large cell), III (large cell) and/or subtype: diffuse and predominantly small cell type), low grade/follicular non-Hodgkin's lymphoma (NHL), intermediate grade/follicular NHL, marginal zone B-cell lymphoma (e.g., extranodal (e.g., MALT-type+/−monocytoid B cells) and/or Nodal (e.g., +/−monocytoid B cells)), splenic marginal zone lymphoma (e.g., +/−villous lymphocytes), Hairy cell leukemia, plasmacytoma/plasma cell myeloma (e.g., myeloma and multiple myeloma), diffuse large B-cell lymphoma (e.g., primary mediastinal (thymic) B-cell lymphoma), intermediate grade diffuse NHL, Burkitt's lymphoma, High-grade B-cell lymphoma, Burkitt-like, high grade immunoblastic NHL, high grade lymphoblastic NHL, high grade small non-cleaved cell NHL, bulky disease NHL, AIDS-related lymphoma, and Waldenstrom's macroglobulinemia).

In some embodiments, the cancer is a T-cell and/or putative NK-cell cancer. In some embodiments, the cancer is precursor T-cell cancer (precursor T-lymphoblastic lymphoma/leukemia) and peripheral T-cell and NK-cell cancers (e.g., T-cell chronic lymphocytic leukemia/prolymphocytic leukemia, and large granular lymphocyte leukemia (LGL) (e.g., T-cell type and/or NK-cell type), cutaneous T-cell lymphoma (e.g., mycosis fungoides/Sezary syndrome), primary T-cell lymphomas unspecified (e.g., cytological categories (e.g., medium-sized cell, mixed medium and large cell), large cell, lymphoepitheloid cell, subtype hepatosplenic γδ T-cell lymphoma, and subcutaneous panniculitic T-cell lymphoma), angioimmunoblastic T-cell lymphoma (AILD), angiocentric lymphoma, intestinal T-cell lymphoma (e.g., +/−enteropathy associated), adult T-cell lymphoma/leukemia (ATL), anaplastic large cell lymphoma (ALCL) (e.g., CD30+, T- and null-cell types), anaplastic large-cell lymphoma, and Hodgkin's like).

In some embodiments, the cancer is Hodgkin's disease.

In some embodiments, the cancer is leukemia. In some embodiments, the cancer is chronic myelocytic I (granulocytic) leukemia, chronic myelogenous, and chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), acute myeloid leukemia, acute lymphocytic leukemia, and acute myelocytic leukemia (e.g., myeloblastic, promyelocytic, myelomonocytic, monocytic, and erythroleukemia).

In some embodiments, the cancer is a liquid tumor or plasmacytoma. In some embodiments, the cancer is extramedullary plasmacytoma, a solitary myeloma, and multiple myeloma. In some embodiments, the plasmacytoma is multiple myeloma.

In some embodiments, the cancer is lung cancer.

In some embodiments, the cancer is prostate cancer. In some embodiments, the prostate cancer is an adenocarcinoma. In some embodiments, the prostate cancer is a sarcoma, neuroendocrine tumor, small cell cancer, ductal cancer, or a lymphoma. In some embodiments, the prostate cancer is stage A prostate cancer (the cancer cannot be felt during a rectal exam). In some embodiments, the prostate cancer is stage B prostate cancer (i.e., the tumor involves more tissue within the prostate, it can be felt during a rectal exam, or it is found with a biopsy that is done because of a high PSA level). In some embodiments, the prostate cancer is stage C prostate cancer (i.e., the cancer has spread outside the prostate to nearby tissues). In some embodiments, the prostate cancer is stage D prostate cancer. In some embodiments, the prostate cancer is androgen independent prostate cancer (AIPC). In some embodiments, the prostate cancer is androgen dependent prostate cancer. In some embodiments, the prostate cancer is refractory to hormone therapy. In some embodiments, the prostate cancer is substantially refractory to hormone therapy. In some embodiments, the prostate cancer is refractory to chemotherapy. In some embodiments, the prostate cancer is metastatic prostate cancer. In some embodiments, the individual is a human who has a gene, genetic mutation, or polymorphism associated with prostate cancer (e.g., RNASEL/HPC1, ELAC2/HPC2, SR-A/MSR1, CHEK2, BRCA2, PON1, OGG1, MIC-1, TLR4, and PTEN) or has one or more extra copies of a gene associated with prostate cancer. In some embodiments, the prostate cancer is HER2 positive. In some embodiments, the prostate cancer is HER2 negative.

In some embodiments, the cancer has metastasized and is characterized by circulating tumor cells.

Imaging Uses

The selective delivery molecules of Formula I allow the targeted delivery of imaging agents to specific cells and/or tissues (e.g., cancerous tissues). The molecules comprise a basic peptide sequence (B) which is designed to be transported across a cellular membrane or retained by tissue, an acidic peptide sequence (A) which inhibits uptake and retention of peptide B into cells, a linker X which is cleavable under specific conditions, imaging moieties bound to peptides A and B, or X and a macromolecular carrier. In some embodiments, cleavage of the linker X linker frees peptide B from peptide A and allows the transport of peptide B (and any imaging moieties attached thereto) across a cellular membrane or retention of B to tissue. In some embodiments, the selective delivery molecules of Formula I enable targeted delivery of one or more imaging agents to a cell or tissue. In some embodiments, targeted delivery of an imaging agent to a cell or tissue enables a medical professional to visualize/image a specific tissue.

Disclosed herein, in certain embodiments, are methods of delivering imaging agents to a tissue of interest, comprising contacting the tissue of interest with a molecule of Formula I: [D_(A)-c _(A)]-A-[c _(M)-M]-X-B-[c _(B)-D_(B)]  Formula I wherein,

-   -   X is a cleavable linker;     -   A is a peptide with a sequence comprising 5 to 9 acidic amino         acids;     -   B is a peptide with a sequence comprising 7 to 9 basic amino         acids;     -   c_(A), c_(B), and c_(M) each independently comprise 0-1 amino         acid;     -   M is a polyethylene glycol (PEG) polymer; and     -   D_(A) and D_(B) are each independently an imaging agent; and     -   wherein [c_(M)-M] is bound to at any position on A or X,         [D_(A)-c_(A)] is bound to any amino acid on A, and [c_(B)-D_(B)]         is bound to any amino acid on B.         In some embodiments, A and B do not have an equal number of         acidic and basic amino acids. In some embodiments, the number of         basic amino acids in B is greater than the number of acidic         amino acids in A. In some embodiments, A is a peptide comprising         5 or 9 consecutive glutamates (SEQ ID NO: 3). In some         embodiments, B is a peptide comprising 8 or 9 consecutive         arginines (SEQ ID NO: 4). In some embodiments, A is a peptide         comprising 5 or 9 consecutive glutamates (SEQ ID NO: 3) and B is         a peptide comprising 8 or 9 consecutive arginines (SEQ ID NO:         4). In some embodiments, A is a peptide comprising 5 consecutive         glutamates (SEQ ID NO: 5) and B is a peptide comprising 8         consecutive arginines (SEQ ID NO: 6). In some embodiments,         c_(A), c_(B), and c_(M) are each independently a 0-1 amino acid.         In some embodiments, c_(A), c_(B), and c_(M) are each         independently selected from a naturally-occurring amino acid or         a non-naturally-occurring amino acid. In some embodiments,         c_(A), c_(B), and c_(M) are each independently selected from a D         amino acid, a L amino acid, an α-amino acid, a β-amino acid, or         a γ-amino acid. In some embodiments, c_(A), c_(B), and c_(M) are         each independently selected from any amino acid having a free         thiol group, any amino acid having a N-terminal amine group, and         any amino acid with a side chain capable of forming an oxime or         hydrazone bond upon reaction with a hydroxylamine or hydrazine         group. In some embodiments, c_(A), c_(B), and c_(M) are each         independently selected from D-cysteine, D-glutamate, lysine, and         para-4-acetyl L-phenylalanine. In some embodiments, c_(B) is any         amino acid having a free thiol group. In some embodiments, c_(B)         is D-cysteine. In some embodiments, c_(A) is any amino acid         having a N-terminal amine group. In some embodiments, c_(A) is         D-glutamate. In some embodiments, c_(A) is lysine. In some         embodiments, c_(M) is any amino acid with a side chain capable         of forming an oxime or hydrazone bond upon reaction with a         hydroxylamine or hydrazine group. In some embodiments, c_(M) is         para-4-acetyl L-phenylalanine. In some embodiments, X is         cleavable by a protease. In some embodiments, X is cleavable by         a matrix metalloproteinase. In some embodiments, X comprises an         amino acid sequence that is cleavable by MMP2, MMP7, MMP9, or         MMP14. In some embodiments, X comprises a peptide linkage. In         some embodiments, X comprises an amino acid sequence selected         from: PLGLAG (SEQ ID NO: 2), PLG-C(me)-AG (SEQ ID NO: 1),         RPLALWRS (SEQ ID NO: 7), ESPAYYTA (SEQ ID NO: 8), DPRSFL (SEQ ID         NO: 9), PPRSFL (SEQ ID NO: 10), RLQLKL (SEQ ID NO: 11), and         RLQLK(Ac) (SEQ ID NO: 12). In some embodiments, X comprises the         amino acid sequence PLGLAG (SEQ ID NO: 2). In some embodiments,         X comprises the amino acid sequence PLG-C(me)-AG (SEQ ID NO: 1).         In some embodiments, X comprises the amino acid sequence         RPLALWRS (SEQ ID NO: 7). In some embodiments, X comprises the         amino acid sequence DPRSFL (SEQ ID NO: 9). In some embodiments,         X comprises the amino acid sequence PPRSFL (SEQ ID NO: 10). In         some embodiments, X comprises the amino acid sequence RLQLKL         (SEQ ID NO: 11). In some embodiments, X comprises the amino acid         sequence RLQLK(Ac) (SEQ ID NO: 12). In some embodiments, D_(A)         and D_(B) are a pair of acceptor and donor fluorescent moieties         that are capable of undergoing Försters/fluorescence resonance         energy transfer with the other. In some embodiments, D_(A) and         D_(B) are Cy5 and Cy7. In some embodiments, D_(A) and D_(B) are         Cy5 and IRDye750. In some embodiments, D_(A) and D_(B) are Cy5         and IRDye800. In some embodiments, D_(A) and D_(B) are Cy5 and         ICG. In some embodiments, D_(A) and D_(B) are a fluorescent         moiety and a fluorescence-quenching moiety.

Disclosed herein, in certain embodiments, are methods of delivering imaging agents to a tissue of interest, comprising contacting the tissue of interest with a molecule of Formula I: [D_(A)-c _(A)]-A-[c _(M)-M]-X-B-[c _(B)-D_(B)]  Formula I wherein,

-   -   X is a peptide linker cleavable by a matrix metalloproteinase;     -   A is a peptide with a sequence comprising 5 or 9 consecutive         glutamates (SEQ ID NO: 3);     -   B is a peptide with a sequence comprising 8 or 9 consecutive         arginines (SEQ ID NO: 4); c_(A), c_(B), and c_(M) each         independently comprise 0-1 amino acid;     -   M is a polyethylene glycol (PEG) polymer; and     -   D_(A) and D_(B) are each independently an imaging agent; and         wherein [c_(M)-M] is bound to at any position on A or X,         [D_(A)-c_(A)] is bound to any amino acid on A, and [c_(B)-D_(B)]         is bound to any amino acid on B.         In some embodiments, A and B do not have an equal number of         acidic and basic amino acids. In some embodiments, c_(A), c_(B),         and c_(M) are each independently a 0-1 amino acid. In some         embodiments, c_(A), c_(B), and c_(M) are each independently         selected from any amino acid having a free thiol group, any         amino acid having a N-terminal amine group, and any amino acid         with a side chain capable of forming an oxime or hydrazone bond         upon reaction with a hydroxylamine or hydrazine group. In some         embodiments, c_(A), c_(B), and c_(M) are each independently         selected from D-cysteine, D-glutamate, lysine, and para-4-acetyl         L-phenylalanine. In some embodiments, c_(B) is any amino acid         having a free thiol group. In some embodiments, c_(B) is         D-cysteine. In some embodiments, c_(A) is any amino acid having         a N-terminal amine group. In some embodiments, c_(A) is         D-glutamate. In some embodiments, c_(M) is any amino acid with a         side chain capable of forming an oxime or hydrazone bond upon         reaction with a hydroxylamine or hydrazine group. In some         embodiments, c_(M) is para-4-acetyl L-phenylalanine. In some         embodiments, X comprises the amino acid sequence RPLALWRS (SEQ         ID NO: 7). In some embodiments, X comprises the amino acid         sequence DPRSFL (SEQ ID NO: 9). In some embodiments, X comprises         the amino acid sequence PPRSFL (SEQ ID NO: 10). In some         embodiments, X comprises the amino acid sequence RLQLKL (SEQ ID         NO: 11). In some embodiments, X comprises the amino acid         sequence RLQLK(Ac) (SEQ ID NO: 12). In some embodiments, D_(A)         and D_(B) are a pair of acceptor and donor fluorescent moieties         that are capable of undergoing Försters/fluorescence resonance         energy transfer with the other. In some embodiments, D_(A) and         D_(B) are Cy5 and Cy7. In some embodiments, D_(A) and D_(B) are         Cy5 and IRDye750. In some embodiments, D_(A) and D_(B) are Cy5         and IRDye800. In some embodiments, D_(A) and D_(B) are Cy5 and         ICG. In some embodiments, D_(A) and D_(B) are a fluorescent         moiety and a fluorescence-quenching moiety.

Disclosed herein, in certain embodiments, are methods of delivering imaging agents to a tissue of interest, comprising contacting the tissue of interest with a molecule of Formula I: [D_(A)-c _(A)]-A-[c _(M)-M]-X-B-[c _(B)-D_(B)]  Formula I wherein,

-   -   X is a peptide linker cleavable by a matrix metalloproteinase;     -   A is a peptide with a sequence comprising 5 consecutive         glutamates (SEQ ID NO: 5);     -   B is a peptide with a sequence comprising 8 consecutive         arginines (SEQ ID NO: 6);     -   c_(A), c_(B), and c_(M) each independently comprise 0-1 amino         acid;     -   M is a polyethylene glycol (PEG) polymer; and     -   D_(A) and D_(B) are each independently an imaging agent; and         wherein [c_(M)-M] is bound to at any position on A or X,         [D_(A)-c_(A)] is bound to any amino acid on A, and [c_(B)-D_(B)]         is bound to any amino acid on B.         In some embodiments, c_(A), c_(B), and c_(M) are each         independently a 0-1 amino acid. In some embodiments, c_(A),         c_(B), and c_(M) are each independently selected from any amino         acid having a free thiol group, any amino acid having a         N-terminal amine group, and any amino acid with a side chain         capable of forming an oxime or hydrazone bond upon reaction with         a hydroxylamine or hydrazine group. In some embodiments, c_(A),         c_(B), and c_(M) are each independently selected from         D-cysteine, D-glutamate, lysine, and para-4-acetyl         L-phenylalanine. In some embodiments, c_(B) is any amino acid         having a free thiol group. In some embodiments, c_(B) is         D-cysteine. In some embodiments, c_(A) is any amino acid having         a N-terminal amine group. In some embodiments, c_(A) is         D-glutamate. In some embodiments, c_(M) is any amino acid with a         side chain capable of forming an oxime or hydrazone bond upon         reaction with a hydroxylamine or hydrazine group. In some         embodiments, c_(M) is para-4-acetyl L-phenylalanine. In some         embodiments, X comprises the amino acid sequence RPLALWRS (SEQ         ID NO: 7). In some embodiments, X comprises the amino acid         sequence DPRSFL (SEQ ID NO: 9). In some embodiments, X comprises         the amino acid sequence PPRSFL (SEQ ID NO: 10). In some         embodiments, X comprises the amino acid sequence RLQLKL (SEQ ID         NO: 11). In some embodiments, X comprises the amino acid         sequence RLQLK(Ac) (SEQ ID NO: 12). In some embodiments, D_(A)         and D_(B) are a pair of acceptor and donor fluorescent moieties         that are capable of undergoing Försters/fluorescence resonance         energy transfer with the other. In some embodiments, D_(A) and         D_(B) are Cy5 and Cy7. In some embodiments, D_(A) and D_(B) are         Cy5 and IRDye750. In some embodiments, D_(A) and D_(B) are Cy5         and IRDye800. In some embodiments, D_(A) and D_(B) are Cy5 and         ICG. In some embodiments, D_(A) and D_(B) are a fluorescent         moiety and a fluorescence-quenching moiety.

Disclosed herein, in certain embodiments, are methods of delivering imaging agents to a tissue of interest, comprising contacting the tissue of interest with a molecule of Formula I: [D_(A)-c _(A)]-A-[c _(M)-M]-X-B-[c _(B)-D_(B)]  Formula I wherein,

-   -   X is a peptide linker cleavable by a matrix metalloproteinase;     -   A is a peptide with a sequence comprising 9 consecutive         glutamates (SEQ ID NO: 13);     -   B is a peptide with a sequence comprising 9 consecutive         arginines (SEQ ID NO: 14);     -   c_(A), c_(B), and c_(M) each independently comprise 0-1 amino         acid;     -   M is a polyethylene glycol (PEG) polymer; and     -   D_(A) and D_(B) are each independently an imaging agent; and         wherein [c_(M)-M] is bound to at any position on A or X,         [D_(A)-c_(A)] is bound to any amino acid on A, and [c_(B)-D_(B)]         is bound to any amino acid on B.         In some embodiments, c_(A), c_(B), and c_(M) are each         independently a 0-1 amino acid. In some embodiments, c_(A),         c_(B), and c_(M) are each independently selected from any amino         acid having a free thiol group, any amino acid having a         N-terminal amine group, and any amino acid with a side chain         capable of forming an oxime or hydrazone bond upon reaction with         a hydroxylamine or hydrazine group. In some embodiments, c_(A),         c_(B), and c_(M) are each independently selected from         D-cysteine, D-glutamate, lysine, and para-4-acetyl         L-phenylalanine. In some embodiments, c_(B) is any amino acid         having a free thiol group. In some embodiments, c_(B) is         D-cysteine. In some embodiments, c_(A) is any amino acid having         a N-terminal amine group. In some embodiments, c_(A) is         D-glutamate. In some embodiments, c_(M) is any amino acid with a         side chain capable of forming an oxime or hydrazone bond upon         reaction with a hydroxylamine or hydrazine group. In some         embodiments, c_(M) is para-4-acetyl L-phenylalanine. In some         embodiments, X comprises the amino acid sequence RPLALWRS (SEQ         ID NO: 7). In some embodiments, X comprises the amino acid         sequence DPRSFL (SEQ ID NO: 9). In some embodiments, X comprises         the amino acid sequence PPRSFL (SEQ ID NO: 10). In some         embodiments, X comprises the amino acid sequence RLQLKL (SEQ ID         NO: 11). In some embodiments, X comprises the amino acid         sequence RLQLK(Ac) (SEQ ID NO: 12). In some embodiments, D_(A)         and D_(B) are a pair of acceptor and donor fluorescent moieties         that are capable of undergoing Försters/fluorescence resonance         energy transfer with the other. In some embodiments, D_(A) and         D_(B) are Cy5 and Cy7. In some embodiments, D_(A) and D_(B) are         Cy5 and IRDye750. In some embodiments, D_(A) and D_(B) are Cy5         and IRDye800. In some embodiments, D_(A) and D_(B) are Cy5 and         ICG. In some embodiments, D_(A) and D_(B) are a fluorescent         moiety and a fluorescence-quenching moiety.

Disclosed herein, in certain embodiments, are methods of delivering a pair of acceptor and donor fluorescent moieties that are capable of undergoing Försters/fluorescence resonance energy transfer with the other to a tissue of interest, comprising contacting the tissue of interest with a molecule of Formula I: [D_(A)-c _(A)]-A-[c _(M)-M]-X-B-[c _(B)-D_(B)]  Formula I wherein,

-   -   X is a cleavable linker;     -   A is a peptide with a sequence comprising 5 to 9 acidic amino         acids;     -   B is a peptide with a sequence comprising 7 to 9 basic amino         acids;     -   c_(A), c_(B), and c_(M) each independently comprise 0-1 amino         acid;     -   M is a polyethylene glycol (PEG) polymer; and     -   D_(A) and D_(B) are a pair of acceptor and donor fluorescent         moieties that are capable of undergoing Försters/fluorescence         resonance energy transfer with the other; and     -   wherein [c_(M)-M] is bound to at any position on A or X,         [D_(A)-c_(A)] is bound to any amino acid on A, and [c_(B)-D_(B)]         is bound to any amino acid on B.         In some embodiments, A and B do not have an equal number of         acidic and basic amino acids. In some embodiments, the number of         basic amino acids in B is greater than the number of acidic         amino acids in A. In some embodiments, A is a peptide comprising         5 or 9 consecutive glutamates (SEQ ID NO: 3). In some         embodiments, B is a peptide comprising 8 or 9 consecutive         arginines (SEQ ID NO: 4). In some embodiments, A is a peptide         comprising 5 or 9 consecutive glutamates (SEQ ID NO: 3) and B is         a peptide comprising 8 or 9 consecutive arginines (SEQ ID NO:         4). In some embodiments, A is a peptide comprising 5 consecutive         glutamates (SEQ ID NO: 5) and B is a peptide comprising 8         consecutive arginines (SEQ ID NO: 6). In some embodiments,         c_(A), c_(B), and c_(M) are each independently a 0-1 amino acid.         In some embodiments, c_(A), c_(B), and c_(M) are each         independently selected from a naturally-occurring amino acid or         a non-naturally-occurring amino acid. In some embodiments,         c_(A), c_(B), and c_(M) are each independently selected from a D         amino acid, a L amino acid, an α-amino acid, a β-amino acid, or         a γ-amino acid. In some embodiments, c_(A), c_(B), and c_(M) are         each independently selected from any amino acid having a free         thiol group, any amino acid having a N-terminal amine group, and         any amino acid with a side chain capable of forming an oxime or         hydrazone bond upon reaction with a hydroxylamine or hydrazine         group. In some embodiments, c_(A), c_(B), and c_(M) are each         independently selected from D-cysteine, D-glutamate, lysine, and         para-4-acetyl L-phenylalanine. In some embodiments, c_(B) is any         amino acid having a free thiol group. In some embodiments, c_(B)         is D-cysteine. In some embodiments, c_(A) is any amino acid         having a N-terminal amine group. In some embodiments, c_(A) is         D-glutamate. In some embodiments, c_(A) is lysine. In some         embodiments, c_(M) is any amino acid with a side chain capable         of forming an oxime or hydrazone bond upon reaction with a         hydroxylamine or hydrazine group. In some embodiments, c_(M) is         para-4-acetyl L-phenylalanine. In some embodiments, X is         cleavable by a protease. In some embodiments, X is cleavable by         a matrix metalloproteinase. In some embodiments, X comprises an         amino acid sequence that is cleavable by MMP2, MMP7, MMP9, or         MMP14. In some embodiments, X comprises a peptide linkage. In         some embodiments, X comprises an amino acid sequence selected         from: PLGLAG (SEQ ID NO: 2), PLG-C(me)-AG (SEQ ID NO: 1),         RPLALWRS (SEQ ID NO: 7), ESPAYYTA (SEQ ID NO: 8), DPRSFL (SEQ ID         NO: 9), PPRSFL (SEQ ID NO: 10), RLQLKL (SEQ ID NO: 11), and         RLQLK(Ac) (SEQ ID NO: 12). In some embodiments, X comprises the         amino acid sequence PLGLAG (SEQ ID NO: 2). In some embodiments,         X comprises the amino acid sequence PLG-C(me)-AG (SEQ ID NO: 1).         In some embodiments, X comprises the amino acid sequence         RPLALWRS (SEQ ID NO: 7). In some embodiments, X comprises the         amino acid sequence DPRSFL (SEQ ID NO: 9). In some embodiments,         X comprises the amino acid sequence PPRSFL (SEQ ID NO: 10). In         some embodiments, X comprises the amino acid sequence RLQLKL         (SEQ ID NO: 11). In some embodiments, X comprises the amino acid         sequence RLQLK(Ac) (SEQ ID NO: 12). In some embodiments, D_(A)         and D_(B) are a pair of acceptor and donor fluorescent moieties         that are capable of undergoing Försters/fluorescence resonance         energy transfer with the other. In some embodiments, D_(A) and         D_(B) are Cy5 and Cy7. In some embodiments, D_(A) and D_(B) are         Cy5 and IRDye750. In some embodiments, D_(A) and D_(B) are Cy5         and IRDye800. In some embodiments, D_(A) and D_(B) are Cy5 and         ICG. In some embodiments, D_(A) and D_(B) are a fluorescent         moiety and a fluorescence-quenching moiety.

Disclosed herein, in certain embodiments, are methods of delivering a pair of acceptor and donor fluorescent moieties that are capable of undergoing Försters/fluorescence resonance energy transfer with the other to a tissue of interest, comprising contacting the tissue of interest with a molecule of Formula I: [D_(A)-c _(A)]-A-[c _(M)-M]-X-B-[c _(B)-D_(B)]  Formula I wherein,

-   -   X is a peptide linker cleavable by a matrix metalloproteinase;     -   A is a peptide with a sequence comprising 5 or 9 consecutive         glutamates (SEQ ID NO: 3);     -   B is a peptide with a sequence comprising 8 or 9 consecutive         arginines (SEQ ID NO: 4);     -   c_(A), c_(B), and c_(M) each independently comprise 0-1 amino         acid;     -   M is a polyethylene glycol (PEG) polymer; and     -   D_(A) and D_(B) are a pair of acceptor and donor fluorescent         moieties that are capable of undergoing Försters/fluorescence         resonance energy transfer with the other; and         wherein [c_(M)-M] is bound to at any position on A or X,         [D_(A)-c_(A)] is bound to any amino acid on A, and [c_(B)-D_(B)]         is bound to any amino acid on B.         In some embodiments, A and B do not have an equal number of         acidic and basic amino acids. In some embodiments, c_(A), c_(B),         and c_(M) are each independently a 0-1 amino acid. In some         embodiments, c_(A), c_(B), and c_(M) are each independently         selected from any amino acid having a free thiol group, any         amino acid having a N-terminal amine group, and any amino acid         with a side chain capable of forming an oxime or hydrazone bond         upon reaction with a hydroxylamine or hydrazine group. In some         embodiments, c_(A), c_(B), and c_(M) are each independently         selected from D-cysteine, D-glutamate, lysine, and para-4-acetyl         L-phenylalanine. In some embodiments, c_(B) is any amino acid         having a free thiol group. In some embodiments, c_(B) is         D-cysteine. In some embodiments, c_(A) is any amino acid having         a N-terminal amine group. In some embodiments, c_(A) is         D-glutamate. In some embodiments, c_(M) is any amino acid with a         side chain capable of forming an oxime or hydrazone bond upon         reaction with a hydroxylamine or hydrazine group. In some         embodiments, c_(M) is para-4-acetyl L-phenylalanine. In some         embodiments, X comprises the amino acid sequence RPLALWRS (SEQ         ID NO: 7). In some embodiments, X comprises the amino acid         sequence DPRSFL (SEQ ID NO: 9). In some embodiments, X comprises         the amino acid sequence PPRSFL (SEQ ID NO: 10). In some         embodiments, X comprises the amino acid sequence RLQLKL (SEQ ID         NO: 11). In some embodiments, X comprises the amino acid         sequence RLQLK(Ac) (SEQ ID NO: 12). In some embodiments, D_(A)         and D_(B) are Cy5 and Cy7. In some embodiments, D_(A) and D_(B)         are Cy5 and Cy7.

Disclosed herein, in certain embodiments, are methods of delivering a pair of acceptor and donor fluorescent moieties that are capable of undergoing Försters/fluorescence resonance energy transfer with the other to a tissue of interest, comprising contacting the tissue of interest with a molecule of Formula I: [D_(A)-c _(A)]-A-[c _(M)-M]-X-B-[c _(B)-D_(B)]  Formula I wherein,

-   -   X is a peptide linker cleavable by a matrix metalloproteinase;     -   A is a peptide with a sequence comprising 5 consecutive         glutamates (SEQ ID NO: 5);     -   B is a peptide with a sequence comprising 8 consecutive         arginines (SEQ ID NO: 6);     -   c_(A), c_(B), and c_(M) each independently comprise 0-1 amino         acid;     -   M is a polyethylene glycol (PEG) polymer; and     -   D_(A) and D_(B) are a pair of acceptor and donor fluorescent         moieties that are capable of undergoing Försters/fluorescence         resonance energy transfer with the other; and         wherein [c_(M)-M] is bound to at any position on A or X,         [D_(A)-c_(A)] is bound to any amino acid on A, and [c_(B)-D_(B)]         is bound to any amino acid on B.         In some embodiments, c_(A), c_(B), and c_(M) are each         independently a 0-1 amino acid. In some embodiments, c_(A),         c_(B), and c_(M) are each independently selected from any amino         acid having a free thiol group, any amino acid having a         N-terminal amine group, and any amino acid with a side chain         capable of forming an oxime or hydrazone bond upon reaction with         a hydroxylamine or hydrazine group. In some embodiments, c_(A),         c_(B), and c_(M) are each independently selected from         D-cysteine, D-glutamate, lysine, and para-4-acetyl         L-phenylalanine. In some embodiments, c_(B) is any amino acid         having a free thiol group. In some embodiments, c_(B) is         D-cysteine. In some embodiments, c_(A) is any amino acid having         a N-terminal amine group. In some embodiments, c_(A) is         D-glutamate. In some embodiments, c_(M) is any amino acid with a         side chain capable of forming an oxime or hydrazone bond upon         reaction with a hydroxylamine or hydrazine group. In some         embodiments, c_(M) is para-4-acetyl L-phenylalanine. In some         embodiments, X comprises the amino acid sequence RPLALWRS (SEQ         ID NO: 7). In some embodiments, X comprises the amino acid         sequence DPRSFL (SEQ ID NO: 9). In some embodiments, X comprises         the amino acid sequence PPRSFL (SEQ ID NO: 10). In some         embodiments, X comprises the amino acid sequence RLQLKL (SEQ ID         NO: 11). In some embodiments, X comprises the amino acid         sequence RLQLK(Ac) (SEQ ID NO: 12). In some embodiments, D_(A)         and D_(B) are Cy5 and Cy7. In some embodiments, D_(A) and D_(B)         are Cy5 and IRDye750. In some embodiments, D_(A) and D_(B) are         Cy5 and IRDye800. In some embodiments, D_(A) and D_(B) are Cy5         and ICG.

Disclosed herein, in certain embodiments, are methods of delivering a pair of acceptor and donor fluorescent moieties that are capable of undergoing Försters/fluorescence resonance energy transfer with the other to a tissue of interest, comprising contacting the tissue of interest with a molecule of Formula I: [D_(A)-c _(A)]-A-[c _(M)-M]-X-B-[c _(B)-D_(B)]  Formula I wherein,

-   -   X is a peptide linker cleavable by a matrix metalloproteinase;     -   A is a peptide with a sequence comprising 9 consecutive         glutamates (SEQ ID NO: 13);     -   B is a peptide with a sequence comprising 9 consecutive         arginines (SEQ ID NO: 14);     -   c_(A), c_(B), and c_(M) each independently comprise 0-1 amino         acid;     -   M is a polyethylene glycol (PEG) polymer; and     -   D_(A) and D_(B) are a pair of acceptor and donor fluorescent         moieties that are capable of undergoing Försters/fluorescence         resonance energy transfer with the other; and         wherein [c_(M)-M] is bound to at any position on A or X,         [D_(A)-c_(A)] is bound to any amino acid on A, and [c_(B)-D_(B)]         is bound to any amino acid on B.         In some embodiments, c_(A), c_(B), and c_(M) are each         independently selected from any amino acid having a free thiol         group, any amino acid having a N-terminal amine group, and any         amino acid with a side chain capable of forming an oxime or         hydrazone bond upon reaction with a hydroxylamine or hydrazine         group. In some embodiments, c_(A), c_(B), and c_(M) are each         independently a 0-1 amino acid. In some embodiments, c_(A),         c_(B), and c_(M) are each independently selected from         D-cysteine, D-glutamate, lysine, and para-4-acetyl         L-phenylalanine. In some embodiments, c_(B) is any amino acid         having a free thiol group. In some embodiments, c_(B) is         D-cysteine. In some embodiments, c_(A) is any amino acid having         a N-terminal amine group. In some embodiments, c_(A) is         D-glutamate. In some embodiments, c_(M) is any amino acid with a         side chain capable of forming an oxime or hydrazone bond upon         reaction with a hydroxylamine or hydrazine group. In some         embodiments, c_(M) is para-4-acetyl L-phenylalanine. In some         embodiments, X comprises the amino acid sequence RPLALWRS (SEQ         ID NO: 7). In some embodiments, X comprises the amino acid         sequence DPRSFL (SEQ ID NO: 9). In some embodiments, X comprises         the amino acid sequence PPRSFL (SEQ ID NO: 10). In some         embodiments, X comprises the amino acid sequence RLQLKL (SEQ ID         NO: 11). In some embodiments, X comprises the amino acid         sequence RLQLK(Ac) (SEQ ID NO: 12). In some embodiments, D_(A)         and D_(B) are Cy5 and Cy7. In some embodiments, D_(A) and D_(B)         are Cy5 and IRDye750. In some embodiments, D_(A) and D_(B) are         Cy5 and IRDye800. In some embodiments, D_(A) and D_(B) are Cy5         and ICG.

Disclosed herein, in certain embodiments, are methods of delivering a pair of acceptor and donor fluorescent moieties that are capable of undergoing Försters/fluorescence resonance energy transfer with the other to a tissue of interest, comprising contacting the tissue of interest with SDM-14.

Disclosed herein, in certain embodiments, are methods of delivering a pair of acceptor and donor fluorescent moieties that are capable of undergoing Försters/fluorescence resonance energy transfer with the other to a tissue of interest, comprising contacting the tissue of interest with SDM-15.

Disclosed herein, in certain embodiments, are methods of delivering a pair of acceptor and donor fluorescent moieties that are capable of undergoing Försters/fluorescence resonance energy transfer with the other to a tissue of interest, comprising contacting the tissue of interest with SDM-23.

Disclosed herein, in certain embodiments, are methods of delivering a pair of acceptor and donor fluorescent moieties that are capable of undergoing Försters/fluorescence resonance energy transfer with the other to a tissue of interest, comprising contacting the tissue of interest with SDM-24.

Disclosed herein, in certain embodiments, are methods of delivering a pair of acceptor and donor fluorescent moieties that are capable of undergoing Försters/fluorescence resonance energy transfer with the other to a tissue of interest, comprising contacting the tissue of interest with SDM-25.

Disclosed herein, in certain embodiments, are methods of delivering a pair of acceptor and donor fluorescent moieties that are capable of undergoing Försters/fluorescence resonance energy transfer with the other to a tissue of interest, comprising contacting the tissue of interest with SDM-26.

Disclosed herein, in certain embodiments, are methods of delivering a pair of acceptor and donor fluorescent moieties that are capable of undergoing Försters/fluorescence resonance energy transfer with the other to a tissue of interest, comprising contacting the tissue of interest with SDM-27.

Disclosed herein, in certain embodiments, are methods of delivering a pair of acceptor and donor fluorescent moieties that are capable of undergoing Försters/fluorescence resonance energy transfer with the other to a tissue of interest, comprising contacting the tissue of interest with SDM-32.

Disclosed herein, in certain embodiments, are methods of delivering a pair of acceptor and donor fluorescent moieties that are capable of undergoing Försters/fluorescence resonance energy transfer with the other to a tissue of interest, comprising contacting the tissue of interest with SDM-35.

Disclosed herein, in certain embodiments, are methods of visualizing a tissue of interest in an individual in need thereof, comprising:

(a) administering to the individual a molecule of Formula I that localizes to the tissue of interest in the individual, [D_(A)-c _(A)]-A-[c _(M)-M]-X-B-[c _(B)-D_(B)];  Formula I wherein,

-   -   X is a cleavable linker;     -   A is a peptide with a sequence comprising 5 to 9 acidic amino         acids;     -   B is a peptide with a sequence comprising 7 to 9 basic amino         acids;     -   c_(A), c_(B), and c_(M) each independently comprise 0-1 amino         acid;     -   M is a polyethylene glycol (PEG) polymer; and     -   D_(A) and D_(B) are each independently an imaging agent; and     -   wherein [c_(M)-M] is bound to at any position on A or X,         [D_(A)-c_(A)] is bound to any amino acid on A, and [c_(B)-D_(B)]         is bound to any amino acid on B; and         (b) visualizing at least one of the imaging agents.         In some embodiments, the tissue is cancerous. In some         embodiments, the cancerous tissue is: breast cancer tissue,         colorectal cancer tissue, squamous cell carcinoma tissue,         prostate cancer tissue, melanoma tissue, or thyroid cancer         tissue. In some embodiments, the cancerous cell or tissue is         breast cancer tissue. In some embodiments, the cancerous cell or         tissue is colon cancer tissue. In some embodiments, the method         further comprises surgically removing the tissue of interest         from the individual. In some embodiments, the surgical margin         surrounding the tissue of interest is decreased. In some         embodiments, the method further comprises preparing a tissue         sample from the removed cell or tissue of interest. In some         embodiments, the method further comprises staging the cancerous         tissue. In some embodiments, A and B do not have an equal number         of acidic and basic amino acids. In some embodiments, the number         of basic amino acids in B is greater than the number of acidic         amino acids in A. In some embodiments, A is a peptide comprising         5 or 9 consecutive glutamates (SEQ ID NO: 3). In some         embodiments, B is a peptide comprising 8 or 9 consecutive         arginines (SEQ ID NO: 4). In some embodiments, A is a peptide         comprising 5 or 9 consecutive glutamates (SEQ ID NO: 3) and B is         a peptide comprising 8 or 9 consecutive arginines (SEQ ID NO:         4). In some embodiments, A is a peptide comprising 5 consecutive         glutamates (SEQ ID NO: 5) and B is a peptide comprising 8         consecutive arginines (SEQ ID NO: 6). In some embodiments,         c_(A), c_(B), and c_(M) are each independently a 0-1 amino acid.         In some embodiments, c_(A), c_(B), and c_(M) are each         independently selected from a naturally-occurring amino acid or         a non-naturally-occurring amino acid. In some embodiments,         c_(A), c_(B), and c_(M) are each independently selected from a D         amino acid, a L amino acid, an α-amino acid, a β-amino acid, or         a γ-amino acid. In some embodiments, c_(A), c_(B), and c_(M) are         each independently selected from any amino acid having a free         thiol group, any amino acid having a N-terminal amine group, and         any amino acid with a side chain capable of forming an oxime or         hydrazone bond upon reaction with a hydroxylamine or hydrazine         group. In some embodiments, c_(A), c_(B), and c_(M) are each         independently selected from D-cysteine, D-glutamate, lysine, and         para-4-acetyl L-phenylalanine. In some embodiments, c_(B) is any         amino acid having a free thiol group. In some embodiments, c_(B)         is D-cysteine. In some embodiments, c_(A) is any amino acid         having a N-terminal amine group. In some embodiments, c_(A) is         D-glutamate. In some embodiments, c_(A) is lysine. In some         embodiments, c_(M) is any amino acid with a side chain capable         of forming an oxime or hydrazone bond upon reaction with a         hydroxylamine or hydrazine group. In some embodiments, c_(M) is         para-4-acetyl L-phenylalanine. In some embodiments, X is         cleavable by a protease. In some embodiments, X is cleavable by         a matrix metalloproteinase. In some embodiments, X comprises an         amino acid sequence that is cleavable by MMP2, MMP7, MMP9, or         MMP14. In some embodiments, X comprises a peptide linkage. In         some embodiments, X comprises an amino acid sequence selected         from: PLGLAG (SEQ ID NO: 2), PLG-C(me)-AG (SEQ ID NO: 1),         RPLALWRS (SEQ ID NO: 7), ESPAYYTA (SEQ ID NO: 8), DPRSFL (SEQ ID         NO: 9), PPRSFL (SEQ ID NO: 10), RLQLKL (SEQ ID NO: 11), and         RLQLK(Ac) (SEQ ID NO: 12). In some embodiments, X comprises the         amino acid sequence PLGLAG (SEQ ID NO: 2). In some embodiments,         X comprises the amino acid sequence PLG-C(me)-AG (SEQ ID NO: 1).         In some embodiments, X comprises the amino acid sequence         RPLALWRS (SEQ ID NO: 7). In some embodiments, X comprises the         amino acid sequence DPRSFL (SEQ ID NO: 9). In some embodiments,         X comprises the amino acid sequence PPRSFL (SEQ ID NO: 10). In         some embodiments, X comprises the amino acid sequence RLQLKL         (SEQ ID NO: 11). In some embodiments, X comprises the amino acid         sequence RLQLK(Ac) (SEQ ID NO: 12). In some embodiments, D_(A)         and D_(B) are a pair of acceptor and donor fluorescent moieties         that are capable of undergoing Försters/fluorescence resonance         energy transfer with the other. In some embodiments, D_(A) and         D_(B) are Cy5 and Cy7. In some embodiments, D_(A) and D_(B) are         Cy5 and IRDye750. In some embodiments, D_(A) and D_(B) are Cy5         and IRDye800. In some embodiments, D_(A) and D_(B) are Cy5 and         ICG. In some embodiments, the method further comprises         visualizing Försters/fluorescence resonance energy transfer         between D_(A) and D_(B). In some embodiments, D_(A) and D_(B)         are a fluorescent moiety and a fluorescence-quenching moiety. In         some embodiments, the molecule is chosen from: SDM-14, SDM-15,         SDM-23, SDM-24, SDM-25, SDM-26, SDM-27, SDM-32, and SDM-35.

Disclosed herein, in certain embodiments, are methods of visualizing a tissue of interest in an individual in need thereof, comprising:

(a) administering to the individual a molecule of Formula I that localizes to the tissue of interest in the individual, [D_(A)-c _(A)]-A-[c _(M)-M]-X-B-[c _(B)-D_(B)]  Formula I wherein,

-   -   X is a peptide linker cleavable by a matrix metalloproteinase;     -   A is a peptide with a sequence comprising 5 or 9 consecutive         glutamates (SEQ ID NO: 3);     -   B is a peptide with a sequence comprising 8 or 9 consecutive         arginines (SEQ ID NO: 4);     -   c_(A), c_(B), and c_(M) each independently comprise 0-1 amino         acid;     -   M is a polyethylene glycol (PEG) polymer; and     -   D_(A) and D_(B) are a pair of acceptor and donor fluorescent         moieties that are capable of undergoing Försters/fluorescence         resonance energy transfer with the other; and     -   wherein [c_(M)-M] is bound to at any position on A or X,         [D_(A)-c_(A)] is bound to any amino acid on A, and [c_(B)-D_(B)]         is bound to any amino acid on B; and         (b) visualizing at least one of the imaging agents.         In some embodiments, A and B do not have an equal number of         acidic and basic amino acids. In some embodiments, c_(A), c_(B),         and c_(M) are each independently a 0-1 amino acid. In some         embodiments, c_(A), c_(B), and c_(M) are each independently         selected from any amino acid having a free thiol group, any         amino acid having a N-terminal amine group, and any amino acid         with a side chain capable of forming an oxime or hydrazone bond         upon reaction with a hydroxylamine or hydrazine group. In some         embodiments, c_(A), c_(B), and c_(M) are each independently         selected from D-cysteine, D-glutamate, lysine, and para-4-acetyl         L-phenylalanine. In some embodiments, c_(B) is any amino acid         having a free thiol group. In some embodiments, c_(B) is         D-cysteine. In some embodiments, c_(A) is any amino acid having         a N-terminal amine group. In some embodiments, c_(A) is         D-glutamate. In some embodiments, c_(M) is any amino acid with a         side chain capable of forming an oxime or hydrazone bond upon         reaction with a hydroxylamine or hydrazine group. In some         embodiments, c_(M) is para-4-acetyl L-phenylalanine. In some         embodiments, X comprises the amino acid sequence RPLALWRS (SEQ         ID NO: 7). In some embodiments, X comprises the amino acid         sequence DPRSFL (SEQ ID NO: 9). In some embodiments, X comprises         the amino acid sequence PPRSFL (SEQ ID NO: 10). In some         embodiments, X comprises the amino acid sequence RLQLKL (SEQ ID         NO: 11). In some embodiments, X comprises the amino acid         sequence RLQLK(Ac) (SEQ ID NO: 12). In some embodiments, D_(A)         and D_(B) are Cy5 and Cy7. In some embodiments, D_(A) and D_(B)         are Cy5 and Cy7.

Disclosed herein, in certain embodiments, are methods of visualizing a tissue of interest in an individual in need thereof, comprising:

(a) administering to the individual a molecule of Formula I that localizes to the tissue of interest in the individual: [D_(A)-c _(A)]-A-[c _(M)-M]-X-B-[c _(B)-D_(B)]  Formula I

-   -   wherein,         -   X is a peptide linker cleavable by a matrix             metalloproteinase;         -   A is a peptide with a sequence comprising 5 consecutive             glutamates (SEQ ID NO: 5);         -   B is a peptide with a sequence comprising 8 consecutive             arginines (SEQ ID NO: 6);         -   c_(A), c_(B), and c_(M) each independently comprise 0-1             amino acid;         -   M is a polyethylene glycol (PEG) polymer; and         -   D_(A) and D_(B) are a pair of acceptor and donor fluorescent             moieties that are capable of undergoing             Försters/fluorescence resonance energy transfer with the             other; and     -   wherein [c_(M)-M] is bound to at any position on A or X,         [D_(A)-c_(A)] is bound to any amino acid on A, and [c_(B)-D_(B)]         is bound to any amino acid on B; and         (b) visualizing at least one of the imaging agents.         In some embodiments, c_(A), c_(B), and c_(M) are each         independently a 0-1 amino acid. In some embodiments, c_(A),         c_(B), and c_(M) are each independently selected from any amino         acid having a free thiol group, any amino acid having a         N-terminal amine group, and any amino acid with a side chain         capable of forming an oxime or hydrazone bond upon reaction with         a hydroxylamine or hydrazine group. In some embodiments, c_(A),         c_(B), and c_(M) are each independently selected from         D-cysteine, D-glutamate, lysine, and para-4-acetyl         L-phenylalanine. In some embodiments, c_(B) is any amino acid         having a free thiol group. In some embodiments, c_(B) is         D-cysteine. In some embodiments, c_(A) is any amino acid having         a N-terminal amine group. In some embodiments, c_(A) is         D-glutamate. In some embodiments, c_(M) is any amino acid with a         side chain capable of forming an oxime or hydrazone bond upon         reaction with a hydroxylamine or hydrazine group. In some         embodiments, c_(M) is para-4-acetyl L-phenylalanine. In some         embodiments, X comprises the amino acid sequence RPLALWRS (SEQ         ID NO: 7). In some embodiments, X comprises the amino acid         sequence DPRSFL (SEQ ID NO: 9). In some embodiments, X comprises         the amino acid sequence PPRSFL (SEQ ID NO: 10). In some         embodiments, X comprises the amino acid sequence RLQLKL (SEQ ID         NO: 11). In some embodiments, X comprises the amino acid         sequence RLQLK(Ac) (SEQ ID NO: 12). In some embodiments, D_(A)         and D_(B) are Cy5 and Cy7. In some embodiments, D_(A) and D_(B)         are Cy5 and IRDye750. In some embodiments, D_(A) and D_(B) are         Cy5 and IRDye800. In some embodiments, D_(A) and D_(B) are Cy5         and ICG.

Disclosed herein, in certain embodiments, are methods of visualizing a tissue of interest in an individual in need thereof, comprising:

(a) administering to the individual a molecule of Formula I that localizes to the tissue of interest in the individual: [D_(A)-c _(A)]-A-[c _(M)-M]-X-B-[c _(B)-D_(B)]  Formula I

-   -   wherein,         -   X is a peptide linker cleavable by a matrix             metalloproteinase;         -   A is a peptide with a sequence comprising 9 consecutive             glutamates (SEQ ID NO: 13);         -   B is a peptide with a sequence comprising 9 consecutive             arginines (SEQ ID NO: 14);         -   c_(A), c_(B), and c_(M) each independently comprise 0-1             amino acid;         -   M is a polyethylene glycol (PEG) polymer; and         -   D_(A) and D_(B) are a pair of acceptor and donor fluorescent             moieties that are capable of undergoing             Försters/fluorescence resonance energy transfer with the             other; and     -   wherein [c_(M)-M] is bound to at any position on A or X,         [D_(A)-c_(A)] is bound to any amino acid on A, and [c_(B)-D_(B)]         is bound to any amino acid on B; and         (b) visualizing at least one of the imaging agents.         In some embodiments, c_(A), c_(B), and c_(M) are each         independently a 0-1 amino acid. In some embodiments, c_(A),         c_(B), and c_(M) are each independently selected from any amino         acid having a free thiol group, any amino acid having a         N-terminal amine group, and any amino acid with a side chain         capable of forming an oxime or hydrazone bond upon reaction with         a hydroxylamine or hydrazine group. In some embodiments, c_(A),         c_(B), and c_(M) are each independently selected from         D-cysteine, D-glutamate, lysine, and para-4-acetyl         L-phenylalanine. In some embodiments, c_(B) is any amino acid         having a free thiol group. In some embodiments, c_(B) is         D-cysteine. In some embodiments, c_(A) is any amino acid having         a N-terminal amine group. In some embodiments, c_(A) is         D-glutamate. In some embodiments, c_(M) is any amino acid with a         side chain capable of forming an oxime or hydrazone bond upon         reaction with a hydroxylamine or hydrazine group. In some         embodiments, c_(M) is para-4-acetyl L-phenylalanine. In some         embodiments, X comprises the amino acid sequence RPLALWRS (SEQ         ID NO: 7). In some embodiments, X comprises the amino acid         sequence DPRSFL (SEQ ID NO: 9). In some embodiments, X comprises         the amino acid sequence PPRSFL (SEQ ID NO: 10). In some         embodiments, X comprises the amino acid sequence RLQLKL (SEQ ID         NO: 11). In some embodiments, X comprises the amino acid         sequence RLQLK(Ac) (SEQ ID NO: 12). In some embodiments, D_(A)         and D_(B) are Cy5 and Cy7. In some embodiments, D_(A) and D_(B)         are Cy5 and IRDye750. In some embodiments, D_(A) and D_(B) are         Cy5 and IRDye800. In some embodiments, D_(A) and D_(B) are Cy5         and ICG.

Disclosed herein, in certain embodiments, are methods of visualizing a tissue of interest in an individual in need thereof, comprising, comprising (a) administering SDM-14 to the individual, and (b) visualizing at least one of the imaging agents.

Disclosed herein, in certain embodiments, are methods of visualizing a tissue of interest in an individual in need thereof, comprising, comprising (a) administering SDM-15 to the individual, and (b) visualizing at least one of the imaging agents.

Disclosed herein, in certain embodiments, are methods of visualizing a tissue of interest in an individual in need thereof, comprising, comprising (a) administering SDM-23 to the individual, and (b) visualizing at least one of the imaging agents.

Disclosed herein, in certain embodiments, are methods of visualizing a tissue of interest in an individual in need thereof, comprising, comprising (a) administering SDM-24 to the individual, and (b) visualizing at least one of the imaging agents.

Disclosed herein, in certain embodiments, are methods of visualizing a tissue of interest in an individual in need thereof, comprising, comprising (a) administering SDM-25 to the individual, and (b) visualizing at least one of the imaging agents.

Disclosed herein, in certain embodiments, are methods of visualizing a tissue of interest in an individual in need thereof, comprising, comprising (a) administering SDM-26 to the individual, and (b) visualizing at least one of the imaging agents.

Disclosed herein, in certain embodiments, are methods of visualizing a tissue of interest in an individual in need thereof, comprising, comprising (a) administering SDM-27 to the individual, and (b) visualizing at least one of the imaging agents.

Disclosed herein, in certain embodiments, are methods of visualizing a tissue of interest in an individual in need thereof, comprising, comprising (a) administering SDM-32 to the individual, and (b) visualizing at least one of the imaging agents.

Disclosed herein, in certain embodiments, are methods of visualizing a tissue of interest in an individual in need thereof, comprising, comprising (a) administering SDM-35 to the individual, and (b) visualizing at least one of the imaging agents.

In some embodiments, targeted delivery of an imaging agent to a cell or tissue enables a medical professional to visualize/image a specific tissue (e.g., cancerous tissue). In some embodiments, targeted delivery of an imaging agent to a cell or tissue enables a medical professional to remove (or, surgically excise) the tissue of interest (e.g., cancerous tissue). In some embodiments, targeted delivery of an imaging agent to a cell or tissue enables a medical professional to remove (or, surgically excise) the tissue of interest (e.g., cancerous tissue) with a decrease in surgical margins. In some embodiments, targeted delivery of an imaging agent to a cell or tissue enables a medical professional to remove (or, surgically excise) a tumor/cancerous tissue and decreases the chance that some of the tumor/cancerous tissue will not be removed. In some embodiments, targeted delivery of an imaging agent to a cell or tissue enables a medical professional to maximally debulk a tumor/cancerous tissue. In some embodiments, targeted delivery of an imaging agent to cancerous breast tissue decreases the chances of an unnecessary operations and re-operations.

In some embodiments, targeted delivery of an imaging agent to a cell or tissue enables a medical professional to more accurately sample (e.g., biopsy (e.g., excision biopsy, incision, biopsy, aspiration biopsy, or needle biopsy)) tissue of interest (e.g., cancerous tissue). In some embodiments, targeted delivery of an imaging agent to a cell or tissue enables a medical professional to visualize/image a specific tissue (e.g., cancerous tissue) within an excised tissue containing healthy tissue. Enabling identification of target tissue (e.g., cancerous tissue) can guide the pathologist on where to section of pathological evaluation and decreases the chances of a pathologist missing unhealthy tissue (e.g., cancerous tissue) and sampling healthy tissue which may produce a false negative. In some embodiments, tissue (e.g., cancerous tissue) removed following use of a compound of Formula I is used to prepare a pathology section or slide. In some embodiments, cancerous tissue removed following use of a compound of Formula I is used to prepare a pathology section or slide which is used to diagnose a tissue as malignant or benign.

In some embodiments, targeted delivery of an imaging agent to cancerous breast tissue enables a medical professional to accurately stage cancer enabling medical treatment decisions. In some embodiments, targeted delivery of an imaging agent to cancerous tissue enables a medical professional to observe the size of a tumor (cancerous tissue) or the spread (e.g., metastatic lesions) of cancerous tissue. In some embodiments, targeted delivery of an imaging agent to a cell or tissue enables a medical professional to design an efficacious treatment regimen.

In some embodiments, a selective delivery molecule according to Formula I comprising an imaging agent is employed in guided surgery. In some embodiments, the selective delivery molecule preferentially localized to cancerous, or other pathological tissues with up-regulated protease activity (e.g. tissues undergoing inflammatory response). In some embodiments, a selective delivery molecule according to Formula I comprising an imaging agent is employed in a guided surgery to remove colorectal cancer. In some embodiments, guided surgery employing the selective delivery molecule allows a surgeon to excise as little healthy (i.e., non-cancerous) tissue as possible. In some embodiments, guided surgery employing the selective delivery molecule allows a surgeon to visualize and excise more cancerous tissue than the surgeon would have been able to excise without the presence of the selective delivery molecule. In some embodiments, the surgery is fluorescence-guided surgery.

Imaging Agents

In some embodiments, an imaging agent is a dye. In some embodiments, an imaging agent is a fluorescent moiety. In some embodiments, a fluorescent moiety is selected from: a fluorescent protein, a fluorescent peptide, a fluorescent dye, a fluorescent material or a combination thereof.

All fluorescent moieties are encompassed within the term “fluorescent moiety.” Specific examples of fluorescent moieties given herein are illustrative and are not meant to limit the fluorescent moieties for use with the targeting molecules disclosed herein.

Examples of fluorescent dyes include, but are not limited to, xanthenes (e.g., rhodamines, rhodols and fluoresceins, and their derivatives); bimanes; coumarins and their derivatives (e.g., umbelliferone and aminomethyl coumarins); aromatic amines (e.g., dansyl; squarate dyes); benzofurans; fluorescent cyanines; indocarbocyanines; carbazoles; dicyanomethylene pyranes; polymethine; oxabenzanthrane; xanthene; pyrylium; carbostyl; perylene; acridone; quinacridone; rubrene; anthracene; coronene; phenanthrecene; pyrene; butadiene; stilbene; porphyrin; pthalocyanine; lanthanide metal chelate complexes; rare-earth metal chelate complexes; and derivatives of such dyes.

Examples of fluorescein dyes include, but are not limited to, 5-carboxyfluorescein, fluorescein-5-isothiocyanate, fluorescein-6-isothiocyanate and 6-carboxyfluorescein.

Examples of rhodamine dyes include, but are not limited to, tetramethylrhodamine-6-isothiocyanate, 5-carboxytetramethylrhodamine, 5-carboxy rhodol derivatives, tetramethyl and tetraethyl rhodamine, diphenyldimethyl and diphenyldiethyl rhodamine, dinaphthyl rhodamine, rhodamine 101 sulfonyl chloride (sold under the tradename of TEXAS RED®).

Examples of cyanine dyes include, but are not limited to, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, Cy7, IRDYE680, Alexa Fluor 750, IRDye800CW, ICG.

Examples of fluorescent peptides include GFP (Green Fluorescent Protein) or derivatives of GFP (e.g., EBFP, EBFP2, Azurite, mKalamal, ECFP, Cerulean, CyPet, YFP, Citrine, Venus, YPet).

Fluorescent labels are detected by any suitable method. For example, a fluorescent label may be detected by exciting the fluorochrome with the appropriate wavelength of light and detecting the resulting fluorescence, e.g., by microscopy, visual inspection, via photographic film, by the use of electronic detectors such as charge coupled devices (CCDs), photomultipliers, etc.

In some embodiments, the imaging agent is labeled with a positron-emitting isotope (e.g., ¹⁸F) for positron emission tomography (PET), gamma-ray isotope (e.g., ^(99m)Tc) for single photon emission computed tomography (SPECT), or a paramagnetic molecule or nanoparticle (e.g., Gd³⁺ chelate or coated magnetite nanoparticle) for magnetic resonance imaging (MRI).

In some embodiments, the imaging agent is labeled with: a gadolinium chelate, an iron oxide particle, a super paramagnetic iron oxide particle, an ultra small paramagnetic particle, a manganese chelate or gallium containing agent.

Examples of gadolinium chelates include, but are not limited to diethylene triamine pentaacetic acid (DTPA), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), and 1,4,7-triazacyclononane-N,N′,N″-triacetic acid (NOTA).

In some embodiments, the imaging agent is a near-infrared fluorophore for near-infra red (near-IR) imaging, a luciferase (firefly, bacterial, or coelenterate) or other luminescent molecule for bioluminescence imaging, or a perfluorocarbon-filled vesicle for ultrasound.

In some embodiments, the imaging agent is a nuclear probe. In some embodiments, the imaging agent is a SPECT or PET radionuclide probe. In some embodiments, the radionuclide probe is selected from: a technetium chelate, a copper chelate, a radioactive fluorine, a radioactive iodine, a indiuim chelate.

Examples of Tc chelates include, but are not limited to HYNIC, DTPA, and DOTA.

In some embodiments, the imaging agent contains a radioactive moiety, for example a radioactive isotope such as ²¹¹At, ¹³¹I, ¹²⁵I, ⁹⁰Y, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁵³Sm, ²¹²Bi, ³²P, ⁶⁴Cu radioactive isotopes of Lu, and others.

Therapeutic Uses

The selective delivery molecules of Formula I allow the targeted delivery of therapeutic agents to specific cells and/or tissues (e.g., cancerous tissues). The molecules comprise a basic peptide sequence (B) which is designed to be transported across a cellular membrane, an acidic peptide sequence (A) which inhibits uptake of peptide B into cells, a linker X which is cleavable under specific conditions, therapeutic agents bound to peptides A and B, or X and a macromolecular carrier. In some embodiments, cleavage of the linker X linker frees peptide B from peptide A and allows the transport of peptide B (and any therapeutic agents attached thereto) across a cellular membrane. In some embodiments, the selective delivery molecules of Formula I enable targeted delivery of one or more therapeutic agents to a cell or tissue. In some embodiments, targeted delivery of a therapeutic agent to a cell or tissue enables a medical professional to treat a specific tissue.

Disclosed herein, in certain embodiments, are methods of delivering a therapeutic agent to a tissue of interest, comprising contacting the tissue of interest with a molecule of Formula I: [D_(A)-c _(A)]-A-[c _(M)-M]-X-B-[c _(B)-D_(B)]  Formula I wherein,

-   -   X is a cleavable linker;     -   A is a peptide with a sequence comprising 5 to 9 acidic amino         acids;     -   B is a peptide with a sequence comprising 7 to 9 basic amino         acids;     -   c_(A), c_(B), and c_(M) each independently comprise 0-1 amino         acid;     -   M is a polyethylene glycol (PEG) polymer; and     -   at least one of D_(A) and D_(B) is independently a therapeutic         agent; and         wherein [c_(M)-M] is bound to at any position on A or X,         [D_(A)-c_(A)] is bound to any amino acid on A, and [c_(B)-D_(B)]         is bound to any amino acid on B.         In some embodiments, A and B do not have an equal number of         acidic and basic amino acids. In some embodiments, the number of         basic amino acids in B is greater than the number of acidic         amino acids in A. In some embodiments, A is a peptide comprising         5 or 9 consecutive glutamates (SEQ ID NO: 3). In some         embodiments, B is a peptide comprising 8 or 9 consecutive         arginines (SEQ ID NO: 4). In some embodiments, A is a peptide         comprising 5 or 9 consecutive glutamates (SEQ ID NO: 3) and B is         a peptide comprising 8 or 9 consecutive arginines (SEQ ID NO:         4). In some embodiments, A is a peptide comprising 5 consecutive         glutamates (SEQ ID NO: 5) and B is a peptide comprising 8         consecutive arginines (SEQ ID NO: 6). In some embodiments,         c_(A), c_(B), and c_(M) are each independently a 0-1 amino acid.         In some embodiments, c_(A), c_(B), and c_(M) are each         independently selected from a naturally-occurring amino acid or         a non-naturally-occurring amino acid. In some embodiments,         c_(A), c_(B), and c_(M) are each independently selected from a D         amino acid, a L amino acid, an α-amino acid, a β-amino acid, or         a γ-amino acid. In some embodiments, c_(A), c_(B), and c_(M) are         each independently selected from any amino acid having a free         thiol group, any amino acid having a N-terminal amine group, and         any amino acid with a side chain capable of forming an oxime or         hydrazone bond upon reaction with a hydroxylamine or hydrazine         group. In some embodiments, c_(A), c_(B), and c_(M) are each         independently selected from D-cysteine, D-glutamate, lysine, and         para-4-acetyl L-phenylalanine. In some embodiments, c_(B) is any         amino acid having a free thiol group. In some embodiments, c_(B)         is D-cysteine. In some embodiments, c_(A) is any amino acid         having a N-terminal amine group. In some embodiments, c_(A) is         D-glutamate. In some embodiments, c_(A) is lysine. In some         embodiments, c_(M) is any amino acid with a side chain capable         of forming an oxime or hydrazone bond upon reaction with a         hydroxylamine or hydrazine group. In some embodiments, c_(M) is         para-4-acetyl L-phenylalanine. In some embodiments, X is         cleavable by a protease. In some embodiments, X is cleavable by         a matrix metalloproteinase. In some embodiments, X comprises an         amino acid sequence that is cleavable by MMP2, MMP7, MMP9, or         MMP14. In some embodiments, X comprises a peptide linkage. In         some embodiments, X comprises an amino acid sequence selected         from: PLGLAG (SEQ ID NO: 2), PLG-C(me)-AG (SEQ ID NO: 1),         RPLALWRS (SEQ ID NO: 7), ESPAYYTA (SEQ ID NO: 8), DPRSFL (SEQ ID         NO: 9), PPRSFL (SEQ ID NO: 10), RLQLKL (SEQ ID NO: 11), and         RLQLK(Ac) (SEQ ID NO: 12). In some embodiments, X comprises the         amino acid sequence PLGLAG (SEQ ID NO: 2). In some embodiments,         X comprises the amino acid sequence PLG-C(me)-AG (SEQ ID NO: 1).         In some embodiments, X comprises the amino acid sequence         RPLALWRS (SEQ ID NO: 7). In some embodiments, X comprises the         amino acid sequence DPRSFL (SEQ ID NO: 9). In some embodiments,         X comprises the amino acid sequence PPRSFL (SEQ ID NO: 10). In         some embodiments, X comprises the amino acid sequence RLQLKL         (SEQ ID NO: 11). In some embodiments, X comprises the amino acid         sequence RLQLK(Ac) (SEQ ID NO: 12).

Disclosed herein, in certain embodiments, are methods of delivering a therapeutic agent to a tissue of interest, comprising contacting the tissue of interest with a molecule of Formula I: [D_(A)-c _(A)]-A-[c _(M)-M]-X-B-[c _(B)-D_(B)]  Formula I wherein,

-   -   X is a peptide linker cleavable by a matrix metalloproteinase;     -   A is a peptide with a sequence comprising 5 or 9 consecutive         glutamates (SEQ ID NO: 3);     -   B is a peptide with a sequence comprising 8 or 9 consecutive         arginines (SEQ ID NO: 4);     -   c_(A), c_(B), and c_(M) each independently comprise 0-1 amino         acid;     -   M is a polyethylene glycol (PEG) polymer; and     -   at least one of D_(A) and D_(B) is independently a therapeutic         agent; and         wherein [c_(M)-M] is bound to at any position on A or X,         [D_(A)-c_(A)] is bound to any amino acid on A, and [c_(B)-D_(B)]         is bound to any amino acid on B.         In some embodiments, A and B do not have an equal number of         acidic and basic amino acids. In some embodiments, c_(A), c_(B),         and c_(M) are each independently a 0-1 amino acid. In some         embodiments, c_(A), c_(B), and c_(M) are each independently         selected from any amino acid having a free thiol group, any         amino acid having a N-terminal amine group, and any amino acid         with a side chain capable of forming an oxime or hydrazone bond         upon reaction with a hydroxylamine or hydrazine group. In some         embodiments, c_(A), c_(B), and c_(M) are each independently         selected from D-cysteine, D-glutamate, lysine, and para-4-acetyl         L-phenylalanine. In some embodiments, c_(B) is any amino acid         having a free thiol group. In some embodiments, c_(B) is         D-cysteine. In some embodiments, c_(A) is any amino acid having         a N-terminal amine group. In some embodiments, c_(A) is         D-glutamate. In some embodiments, c_(M) is any amino acid with a         side chain capable of forming an oxime or hydrazone bond upon         reaction with a hydroxylamine or hydrazine group. In some         embodiments, c_(M) is para-4-acetyl L-phenylalanine. In some         embodiments, X comprises the amino acid sequence PLGLAG (SEQ ID         NO: 2). In some embodiments, X comprises the amino acid sequence         PLG-C(me)-AG (SEQ ID NO: 1).

Disclosed herein, in certain embodiments, are methods of delivering a therapeutic agent to a tissue of interest, comprising contacting the tissue of interest with a molecule of Formula I: [D_(A)-c _(A)]-A-[c _(M)-M]-X-B-[c _(B)-D_(B)]  Formula I wherein,

-   -   X is a peptide linker cleavable by a matrix metalloproteinase;     -   A is a peptide with a sequence comprising 5 consecutive         glutamates (SEQ ID NO: 5);     -   B is a peptide with a sequence comprising 8 consecutive         arginines (SEQ ID NO: 6);     -   c_(A), c_(B), and c_(M) each independently comprise 0-1 amino         acid;     -   M is a polyethylene glycol (PEG) polymer; and     -   at least one of D_(A) and D_(B) is independently a therapeutic         agent; and         wherein [c_(M)-M] is bound to at any position on A or X,         [D_(A)-c_(A)] is bound to any amino acid on A, and [c_(B)-D_(B)]         is bound to any amino acid on B.         In some embodiments, c_(A), c_(B), and c_(M) are each         independently a 0-1 amino acid. In some embodiments, c_(A),         c_(B), and c_(M) are each independently selected from any amino         acid having a free thiol group, any amino acid having a         N-terminal amine group, and any amino acid with a side chain         capable of forming an oxime or hydrazone bond upon reaction with         a hydroxylamine or hydrazine group. In some embodiments, c_(A),         c_(B), and c_(M) are each independently selected from         D-cysteine, D-glutamate, lysine, and para-4-acetyl         L-phenylalanine. In some embodiments, c_(B) is any amino acid         having a free thiol group. In some embodiments, c_(B) is         D-cysteine. In some embodiments, c_(A) is any amino acid having         a N-terminal amine group. In some embodiments, c_(A) is         D-glutamate. In some embodiments, c_(M) is any amino acid with a         side chain capable of forming an oxime or hydrazone bond upon         reaction with a hydroxylamine or hydrazine group. In some         embodiments, c_(M) is para-4-acetyl L-phenylalanine. In some         embodiments, X comprises the amino acid sequence PLGLAG (SEQ ID         NO: 2). In some embodiments, X comprises the amino acid sequence         PLG-C(me)-AG (SEQ ID NO: 1). In some embodiments, X comprises         the amino acid sequence RPLALWRS (SEQ ID NO: 7).

Disclosed herein, in certain embodiments, are methods of delivering a therapeutic agent to a tissue of interest, comprising contacting the tissue of interest with a molecule of Formula I: [D_(A)-c _(A)]-A-[c _(M)-M]-X-B-[c _(B)-D_(B)]  Formula I wherein,

-   -   X is a peptide linker cleavable by a matrix metalloproteinase;     -   A is a peptide with a sequence comprising 9 consecutive         glutamates (SEQ ID NO: 13);     -   B is a peptide with a sequence comprising 9 consecutive         arginines (SEQ ID NO: 14);     -   c_(A), c_(B), and c_(M) each independently comprise 0-1 amino         acid;     -   M is a polyethylene glycol (PEG) polymer; and     -   at least one of D_(A) and D_(B) is independently a therapeutic         agent; and         wherein [c_(M)-M] is bound to at any position on A or X,         [D_(A)-c_(A)] is bound to any amino acid on A, and [c_(B)-D_(B)]         is bound to any amino acid on B.         In some embodiments, c_(A), c_(B), and c_(M) are each         independently a 0-1 amino acid. In some embodiments, c_(A),         c_(B), and c_(M) are each independently selected from any amino         acid having a free thiol group, any amino acid having a         N-terminal amine group, and any amino acid with a side chain         capable of forming an oxime or hydrazone bond upon reaction with         a hydroxylamine or hydrazine group. In some embodiments, c_(A),         c_(B), and c_(M) are each independently selected from         D-cysteine, D-glutamate, lysine, and para-4-acetyl         L-phenylalanine. In some embodiments, c_(B) is any amino acid         having a free thiol group. In some embodiments, c_(B) is         D-cysteine. In some embodiments, c_(A) is any amino acid having         a N-terminal amine group. In some embodiments, c_(A) is         D-glutamate. In some embodiments, c_(M) is any amino acid with a         side chain capable of forming an oxime or hydrazone bond upon         reaction with a hydroxylamine or hydrazine group. In some         embodiments, c_(M) is para-4-acetyl L-phenylalanine. In some         embodiments, X comprises the amino acid sequence PLGLAG (SEQ ID         NO: 2). In some embodiments, X comprises the amino acid sequence         PLG-C(me)-AG (SEQ ID NO: 1). In some embodiments, X comprises         the amino acid sequence RPLALWRS (SEQ ID NO: 7).

In some embodiments, targeted delivery of a therapeutic agent to a cell or tissue enables a medical professional to treat a specific tissue (e.g., cancerous tissue). In some embodiments, targeted delivery of a therapeutic agent to a cell or tissue decreases the dosage of the therapeutic agent. In some embodiments, targeted delivery of a therapeutic agent to a cell or tissue decreases contact of the theraperutic agent with healthy tissue. In some embodiments, targeted delivery of a therapeutic agent to a cell or tissue decreases unwanted side-effects arising from use of high concentrations of a therapeutic agent or contact. In some embodiments, targeted delivery of a therapeutic agent to a cell or tissue decreases unwanted side-effects arising from contact between the therapeutic agent and healthy tissue.

Therapeutic Agents

In some embodiments, a therapeutic agent is selected from: a chemotherapeutic agent, a steroid, an immunotherapeutic agent, a targeted therapy, an anti-inflammatory agent, or a combination thereof.

In some embodiments, a therapeutic agent is a B cell receptor pathway inhibitor. In some embodiments, a therapeutic agent is a CD79A inhibitor, a CD79B inhibitor, a CD19 inhibitor, a Lyn inhibitor, a Syk inhibitor, a PI3K inhibitor, a Blnk inhibitor, a PLCγ inhibitor, a PKCβ inhibitor, or a combination thereof. In some embodiments, a therapeutic agent is an antibody, B cell receptor signaling inhibitor, a PI3K inhibitor, an IAP inhibitor, an mTOR inhibitor, a radioimmunotherapeutic, a DNA damaging agent, a proteosome inhibitor, a histone deacytlase inhibitor, a protein kinase inhibitor, a hedgehog inhibitor, an Hsp90 inhibitor, a telomerase inhibitor, a Jak1/2 inhibitor, a protease inhibitor, a PKC inhibitor, a PARP inhibitor, or a combination thereof. In some embodiments, a therapeutic agent is selected from: chlorambucil, ifosphamide, doxorubicin, mesalazine, thalidomide, lenalidomide, temsirolimus, everolimus, fludarabine, fostamatinib, paclitaxel, docetaxel, ofatumumab, rituximab, dexamethasone, prednisone, CAL-101, ibritumomab, tositumomab, bortezomib, pentostatin, endostatin, bendamustine, chlorambucil, chlormethine, cyclophosphamide, ifosfamide, melphalan, prednimustine, trofosfamide, busulfan, mannosulfan, treosulfan, carboquone, thiotepa, triaziquone, carmustine, fotemustine, lomustine, nimustine, ranimustine, semustine, streptozocin, etoglucid, dacarbazine, mitobronitol, pipobroman, temozolomide, methotrexate, permetrexed, pralatrexate, raltitrexed, cladribine, clofarabine, fludarabine, mercaptopurine, nelarabine, tioguanine, azacitidine, capecitabine, carmofur, cytarabine, decitabine, fluorouracil, gemcitabine, tegafur, vinblastine, vincristine, vindesine, vinflunine, vinorelbine, etopo side, teniposide, demecolcine, docetaxel, paclitaxel, paclitaxel poliglumex, trabectedin, dactinomycin, aclarubicin, daunorubicin, doxorubicin, epirubicin, idarubicin, mitoxantrone, pirarubicin, valrubicin, zorubincin, bleomycin, ixabepilone, mitomycin, plicamycin, carboplatin, cisplatin, oxaliplatin, satraplatin, procarbazine, aminolevulinic acid, efaproxiral, methyl aminolevulinate, porfimer sodium, temoporfin, dasatinib, erlotinib, everolimus, gefitinib, imatinib, lapatinib, nilotinib, pazonanib, sorafenib, sunitinib, temsirolimus, alitretinoin, altretamine, amzacrine, anagrelide, arsenic trioxide, asparaginase, bexarotene, bortezomib, celecoxib, denileukin diftitox, estramustine, hydroxycarbamide, irinotecan, lonidamine, masoprocol, miltefosein, mitoguazone, mitotane, oblimersen, pegaspargase, pentostatin, romidepsin, sitimagene ceradenovec, tiazofurine, topotecan, tretinoin, vorinostat, diethylstilbenol, ethinylestradiol, fosfestrol, polyestradiol phosphate, gestonorone, medroxyprogesterone, megestrol, buserelin, go serelin, leuprorelin, triptorelin, fulvestrant, tamoxifen, toremifene, bicalutamide, flutamide, nilutamide, aminoglutethimide, anastrozole, exemestane, formestane, letrozole, vorozole, abarelix, degarelix, histamine dihydrochloride, mifamurtide, pidotimod, plerixafor, roquinimex, thymopentin, everolimus, gusperimus, leflunomide, mycophenolic acid, sirolimus, ciclosporin, tacrolimus, azathioprine, lenalidomide, methotrexate, thalidomide, iobenguane, ancestim, filgrastim, lenograstim, molgramostim, pegfilgrastim, sargramostim, interferon alfa natural, interferon alfa-2a, interferon alfa-2b, interferon alfacon-1, interferon alfa-n1, interferon beta natural, interferon beta-1a, interferon beta-1b, interferon gamma, peginterferon alfa-2a, peginterferon alfa-2b, aldesleukin, oprelvekin, BCG vaccine, glatiramer acetate, histamine dihydrochloride, immunocyanin, lentinan, melanoma vaccine, mifamurtide, pegademase, pidotimod, plerixafor, poly I:C, poly ICLC, roquinimex, tasonermin, thymopentin, abatacept, abetimus, alefacept, antilymphocyte immunoglobulin (horse), antithymocyte immunoglobulin (rabbit), eculizumab, efalizumab, everolimus, gusperimus, leflunomide, muromab-CD3, mycophenolic acid, natalizumab, sirolimus, adalimumab, afelimomab, certolizumab pegol, etanercept, golimumab, infliximab, anakinra, basiliximab, canakinumab, daclizumab, mepolizumab, rilonacept, tocilizumab, ustekinumab, ciclosporin, tacrolimus, azathioprine, lenalidomide, methotrexate, thalidomide, adalimumab, alemtuzumab, bevacizumab, cetuximab, certolizumab pegol, eculizumab, efalizumab, gemtuzumab, ibritumomab tiuxetan, muromonab-CD3, natalizumab, panitumumab, ranibizumab, rituximab, tositumomab, trastuzumab, catumaxomab, edrecolomab, ofatumumab, muromab-CD3, afelimomab, golimumab, ibritumomab tiuxetan, abagovomab, adecatumumab, alemtuzumab, anti-CD30 monoclonal antibody Xmab2513, anti-MET monoclonal antibody MetMab, apolizumab, apomab, arcitumomab, bispecific antibody 2B1, blinatumomab, brentuximab vedotin, capromab pendetide, cixutumumab, claudiximab, conatumumab, dacetuzumab, denosumab, eculizumab, epratuzumab, epratuzumab, ertumaxomab, etaracizumab, figitumumab, fresolimumab, galiximab, ganitumab, gemtuzumab ozogamicin, glembatumumab, ibritumomab, inotuzumab ozogamicin, ipilimumab, lexatumumab, lintuzumab, lintuzumab, lucatumumab, mapatumumab, matuzumab, milatuzumab, monoclonal antibody CC49, necitumumab, nimotuzumab, ofatumumab, oregovomab, pertuzumab, ramacurimab, ranibizumab, siplizumab, sonepcizumab, tanezumab, tositumomab, trastuzumab, tremelimumab, tucotuzumab celmoleukin, veltuzumab, visilizumab, volociximab, zalutumumab, a syk inhibitor (e.g., R788), enzastaurin, dasatinib, erlotinib, everolimus, gefitinib, imatinib, lapatinib, nilotinib, pazonanib, sorafenib, sunitinib, temsirolimus, an angiogenesis inhibitor (e.g., GT-111, JI-101, R1530), a kinase inhibitors (e.g., AC220, AC480, ACE-041, AMG 900, AP24534, Any-614, AT7519, AT9283, AV-951, axitinib, AZD1152, AZD7762, AZD8055, AZD8931, bafetinib, BAY 73-4506, BGJ398, BGT226, BI 811283, BI6727, BIBF 1120, BIBW 2992, BMS-690154, BMS-777607, BMS-863233, BSK-461364, CAL-101, CEP-11981, CYC116, DCC-2036, dinaciclib, dovitinib lactate, E7050, EMD 1214063, ENMD-2076, fostamatinib disodium, GSK2256098, GSK690693, INCB18424, INNO-406, JNJ-26483327, JX-594, KX2-391, linifanib, LY2603618, MGCD265, MK-0457, MK1496, MLN8054, MLN8237, MP470, NMS-1116354, NMS-1286937, ON 01919.Na, OSI-027, OSI-930, Btk inhibitor, PF-00562271, PF-02341066, PF-03814735, PF-04217903, PF-04554878, PF-04691502, PF-3758309, PHA-739358, PLC3397, progenipoietin, R547, R763, ramucirumab, regorafenib, R05185426, SAR103168, S3333333CH 727965, SGI-1176, SGX523, SNS-314, TAK-593, TAK-901, TKI258, TLN-232, TTP607, XL147, XL228, XL281R05126766, XL418, XL765), an inhibitor of mitogen-activated protein kinase signaling (e.g., U0126, PD98059, PD184352, PD0325901, ARRY-142886, SB239063, SP600125, BAY 43-9006, wortmannin, or LY294002), adriamycin, dactinomycin, bleomycin, vinblastine, cisplatin, acivicin, aclarubicin, acodazole hydrochloride, acronine, adozelesin, aldesleukin, altretamine, ambomycin, ametantrone acetate, aminoglutethimide, amsacrine, anastrozole, anthramycin, asparaginase, asperlin, azacitidine, azetepa, azotomycin, batimastat, benzodepa, bicalutamide, bisantrene hydrochloride, bisnafide dimesylate, bizelesin, bleomycin sulfate, brequinar sodium, bropirimine, busulfan, cactinomycin, calusterone, caracemide, carbetimer, carboplatin, carmustine, carubicin hydrochloride, carzelesin, cedefingol, chlorambucil, cirolemycin, cladribine, crisnatol mesylate, cyclophosphamide, cytarabine, dacarbazine, daunorubicin hydrochloride, decitabine, dexormaplatin, dezaguanine, dezaguanine mesylate, diaziquone, doxorubicin, doxorubicin hydrochloride, droloxifene, droloxifene citrate, dromostanolone propionate, duazomycin, edatrexate, eflornithine hydrochloride, elsamitrucin, enloplatin, enpromate, epipropidine, epirubicin hydrochloride, erbulozole, esorubicin hydrochloride, estramustine, estramustine phosphate sodium, etanidazole, etopo side, etopo side phosphate, etoprine, fadrozole hydrochloride, fazarabine, fenretinide, floxuridine, fludarabine phosphate, fluorouracil, flurocitabine, fosquidone, fostriecin sodium, gemcitabine, gemcitabine hydrochloride, hydroxyurea, idarubicin hydrochloride, ifosfamide, iimofo sine, interleukin II (including recombinant interleukin II, or r1L2), interferon alfa-2a, interferon alfa-2b, interferon alfa-n1, interferon alfa-n3, interferon beta-1 a, interferon gamma-1 b, iproplatin, irinotecan hydrochloride, lanreotide acetate, letrozole, leuprolide acetate, liarozole hydrochloride, lometrexol sodium, lomustine, losoxantrone hydrochloride, masoprocol, maytansine, mechlorethamine hydrochloride, megestrol acetate, melengestrol acetate, melphalan, menogaril, mercaptopurine, methotrexate, methotrexate sodium, metoprine, meturedepa, mitindomide, mitocarcin, mitocromin, mitogillin, mitomalcin, mitomycin, mitosper, mitotane, mitoxantrone hydrochloride, mycophenolic acid, nocodazoie, nogalamycin, ormaplatin, oxisuran, pegaspargase, peliomycin, pentamustine, peplomycin sulfate, perfosfamide, pipobroman, piposulfan, piroxantrone hydrochloride, plicamycin, plomestane, porfimer sodium, porfiromycin, prednimustine, procarbazine hydrochloride, puromycin, puromycin hydrochloride, pyrazofurin, riboprine, rogletimide, safingol, safingol hydrochloride, semustine, simtrazene, sparfosate sodium, sparsomycin, spirogermanium hydrochloride, spiromustine, spiroplatin, streptonigrin, streptozocin, sulofenur, talisomycin, tecogalan sodium, tegafur, teloxantrone hydrochloride, temoporfin, tenipo side, teroxirone, testolactone, thiamiprine, thioguanine, thiotepa, tiazofurin, tirapazamine, toremifene citrate, trestolone acetate, triciribine phosphate, trimetrexate, trimetrexate glucuronate, triptorelin, tubulozole hydrochloride, uracil mustard, uredepa, vapreotide, verteporfin, vinblastine sulfate, vincristine sulfate, vindesine, vindesine sulfate, vinepidine sulfate, vinglycinate sulfate, vinleurosine sulfate, vinorelbine tartrate, vinrosidine sulfate, vinzolidine sulfate, vorozole, zeniplatin, zinostatin, zorubicin hydrochloride. In some embodiments, a therapeutic agent is selected from: 20-epi-1, 25 dihydroxyvitamin D3, 5-ethynyluracil, abiraterone, aclarubicin, acylfulvene, adecypenol, adozelesin, aldesleukin, ALL-TK antagonists, altretamine, ambamustine, amidox, amifostine, aminolevulinic acid, amrubicin, amsacrine, anagrelide, anastrozole, andrographolide, angiogenesis inhibitors, antagonist D, antagonist G, antarelix, anti-dorsalizing morphogenetic protein-1, antiandrogen, prostatic carcinoma, antiestrogen, antineoplaston, antisense oligonucleotides, aphidicolin glycinate, apoptosis gene modulators, apoptosis regulators, apurinic acid, ara-CDP-DL-PTBA, arginine deaminase, asulacrine, atamestane, atrimustine, axinastatin 1, axinastatin 2, axinastatin 3, azasetron, azatoxin, azatyrosine, baccatin III derivatives, balanol, batimastat, BCR/ABL antagonists, benzochlorins, benzoylstaurosporine, beta lactam derivatives, beta-alethine, betaclamycin B, betulinic acid, bFGF inhibitor, bicalutamide, bisantrene, bisaziridinylspermine, bisnafide, bistratene A, bizelesin, breflate, bropirimine, budotitane, buthionine sulfoximine, calcipotriol, calphostin C, camptothecin derivatives, canarypox IL-2, capecitabine, carboxamide-amino-triazole, carboxyamidotriazole, CaRest M3, CARN 700, cartilage derived inhibitor, carzelesin, casein kinase inhibitors (ICOS), castanospermine, cecropin B, cetrorelix, chlorins, chloroquinoxaline sulfonamide, cicaprost, cis-porphyrin, cladribine, clomifene analogues, clotrimazole, collismycin A, collismycin B, combretastatin A4, combretastatin analogue, conagenin, crambescidin 816, crisnatol, cryptophycin 8, cryptophycin A derivatives, curacin A, cyclopentanthraquinones, cycloplatam, cypemycin, cytarabine ocfosfate, cytolytic factor, cytostatin, dacliximab, decitabine, dehydrodidemnin B, deslorelin, dexamethasone, dexifosfamide, dexrazoxane, dexverapamil, diaziquone, didemnin B, didox, diethylnorspermine, dihydro-5-azacytidine, 9-dioxamycin, diphenyl spiromustine, docosanol, dolasetron, doxifluridine, droloxifene, dronabinol, duocarmycin SA, ebselen, ecomustine, edelfosine, edrecolomab, eflornithine, elemene, emitefur, epirubicin, epristeride, estramustine analogue, estrogen agonists, estrogen antagonists, etanidazole, etoposide phosphate, exemestane, fadrozole, fazarabine, fenretinide, filgrastim, finasteride, flavopiridol, flezelastine, fluasterone, fludarabine, fluorodaunorunicin hydrochloride, forfenimex, formestane, fostriecin, fotemustine, gadolinium texaphyrin, gallium nitrate, galocitabine, ganirelix, gelatinase inhibitors, gemcitabine, glutathione inhibitors, hepsulfam, heregulin, hexamethylene bisacetamide, hypericin, ibandronic acid, idarubicin, idoxifene, idramantone, ilmofosine, ilomastat, imidazoacridones, imiquimod, immunostimulant peptides, insulin-such as for example growth factor-1 receptor inhibitor, interferon agonists, interferons, interleukins, iobenguane, iododoxorubicin, ipomeanol, 4-, iroplact, irsogladine, isobengazole, isohomohalicondrin B, itasetron, jasplakinolide, kahalalide F, lamellarin-N triacetate, lanreotide, leinamycin, lenograstim, lentinan sulfate, leptolstatin, letrozole, leukemia inhibiting factor, leukocyte alpha interferon, leuprolide+estrogen+progesterone, leuprorelin, levamisole, liarozole, linear polyamine analogue, lipophilic disaccharide peptide, lipophilic platinum compounds, lissoclinamide 7, lobaplatin, lombricine, lometrexol, lonidamine, losoxantrone, lovastatin, loxoribine, lurtotecan, lutetium texaphyrin, lysofylline, lytic peptides, maitansine, mannostatin A, marimastat, masoprocol, maspin, matrilysin inhibitors, matrix metalloproteinase inhibitors, menogaril, merbarone, meterelin, methioninase, metoclopramide, MIF inhibitor, mifepristone, miltefo sine, mirimostim, mismatched double stranded RNA, mitoguazone, mitolactol, mitomycin analogues, mitonafide, mitotoxin fibroblast growth factor-saporin, mitoxantrone, mofarotene, molgramostim, monoclonal antibody, human chorionic gonadotrophin, monophosphoryl lipid A+myobacterium cell wall sk, mopidamol, multiple drug resistance gene inhibitor, multiple tumor suppressor 1-based therapy, mustard anticancer agent, mycaperoxide B, mycobacterial cell wall extract, myriaporone, N-acetyldinaline, N-substituted benzamides, nafarelin, nagrestip, naloxone+pentazocine, napavin, naphterpin, nartograstim, nedaplatin, nemorubicin, neridronic acid, neutral endopeptidase, nilutamide, nisamycin, nitric oxide modulators, nitroxide antioxidant, nitrullyn, 06-benzylguanine, octreotide, okicenone, oligonucleotides, onapristone, ondansetron, ondansetron, oracin, oral cytokine inducer, ormaplatin, osaterone, oxaliplatin, oxaunomycin, palauamine, palmitoylrhizoxin, pamidronic acid, panaxytriol, panomifene, parabactin, pazelliptine, pegaspargase, peldesine, pentosan polysulfate sodium, pentostatin, pentrozole, perflubron, perfosfamide, perillyl alcohol, phenazinomycin, phenylacetate, phosphatase inhibitors, picibanil, pilocarpine hydrochloride, pirarubicin, piritrexim, placetin A, placetin B, plasminogen activator inhibitor, platinum complex, platinum compounds, platinum-triamine complex, porfimer sodium, porfiromycin, prednisone, propyl bis-acridone, prostaglandin J2, proteasome inhibitors, protein A-based immune modulator, protein kinase C inhibitor, protein kinase C inhibitors, microalgal, protein tyrosine phosphatase inhibitors, purine nucleoside phosphorylase inhibitors, purpurins, pyrazoloacridine, pyridoxylated hemoglobin polyoxyethylerie conjugate, raf antagonists, raltitrexed, ramosetron, ras farnesyl protein transferase inhibitors, ras inhibitors, ras-GAP inhibitor, retelliptine demethylated, rhenium Re 186 etidronate, rhizoxin, ribozymes, RII retinamide, rogletimide, rohitukine, romurtide, roquinimex, rubiginone B1, ruboxyl, safingol, saintopin, SarCNU, sarcophytol A, sargramostim, Sdi 1 mimetics, semustine, senescence derived inhibitor 1, sense oligonucleotides, signal transduction inhibitors, signal transduction modulators, single chain antigen-binding protein, sizofiran, sobuzoxane, sodium borocaptate, sodium phenylacetate, solverol, somatomedin binding protein, sonermin, sparfosic acid, spicamycin D, spiromustine, splenopentin, spongistatin 1, squalamine, stem cell inhibitor, stem-cell division inhibitors, stipiamide, stromelysin inhibitors, sulfino sine, superactive vasoactive intestinal peptide antagonist, suradista, suramin, swainsonine, synthetic glycosaminoglycans, tallimustine, tamoxifen methiodide, tauromustine, tazarotene, tecogalan sodium, tegafur, tellurapyrylium, telomerase inhibitors, temoporfin, temozolomide, tenipo side, tetrachlorodecaoxide, tetrazomine, thaliblastine, thiocoraline, thrombopoietin, thrombopoietin mimetic, thymalfasin, thymopoietin receptor agonist, thymotrinan, thyroid stimulating hormone, tin ethyl etiopurpurin, tirapazamine, titanocene bichloride, top sentin, toremifene, totipotent stem cell factor, translation inhibitors, tretinoin, triacetyluridine, triciribine, trimetrexate, triptorelin, tropisetron, turosteride, tyrosine kinase inhibitors, tyrphostins, UBC inhibitors, ubenimex, urogenital sinus-derived growth inhibitory factor, urokinase receptor antagonists, vapreotide, variolin B, vector system, erythrocyte gene therapy, velaresol, veramine, verdins, verteporfin, vinorelbine, vinxaltine, vitaxin, vorozole, zanoterone, zeniplatin, zilascorb, zinostatin stimalamer, mechloroethamine, cyclophosphamide, chlorambucil, busulfan, carmustine, lomusitne, decarbazine, methotrexate, cytarabine, mercaptopurine, thioguanine, pentostatin, mechloroethamine, cyclophosphamide, chlorambucil, meiphalan, ethylenimine, methylmelamine, hexamethlymelamine, thiotepa, busulfan, carmustine, lomusitne, semustine, streptozocin, decarbazine, fluorouracil, floxouridine, cytarabine, mercaptopurine, thioguanine, pentostatin, erbulozole (also known as R-55104), Dolastatin 10 (also known as DLS-10 and NSC-376128), Mivobulin isethionate (also known as CI-980), Vincristine, NSC-639829, Discodermolide (also known as NVP-XX-A-296), ABT-751 (Abbott, also known as E-7010), Altorhyrtins (such as Altorhyrtin A and Altorhyrtin C), Spongistatins (such as Spongistatin 1, Spongistatin 2, Spongistatin 3, Spongistatin 4, Spongistatin 5, Spongistatin 6, Spongistatin 7, Spongistatin 8, and Spongistatin 9), Cemadotin hydrochloride (also known as LU-103793 and NSC-D-669356), Epothilones (such as Epothilone A, Epothilone B, Epothilone C (also known as desoxyepothilone A or dEpoA), Epothilone D (also referred to as KOS-862, dEpoB, and desoxyepothilone B), Epothilone E, Epothilone F, Epothilone B N-oxide, Epothilone A N-oxide, 16-aza-epothilone B, 21-aminoepothilone B (also known as BMS-310705), 21-hydroxyepothilone D (also known as Desoxyepothilone F and dEpoF), 26-fluoroepothilone), Auristatin PE (also known as NSC-654663), Soblidotin (also known as TZT-1027), LS-4559-P (Pharmacia, also known as LS-4577), LS-4578 (Pharmacia, also known as LS-477-P), LS-4477 (Pharmacia), LS-4559 (Pharmacia), RPR-112378 (Aventis), Vincristine sulfate, DZ-3358 (Daiichi), FR-182877 (Fujisawa, also known as WS-9885B), GS-164 (Takeda), GS-198 (Takeda), KAR-2 (Hungarian Academy of Sciences), BSF-223651 (BASF, also known as ILX-651 and LU-223651), SAH-49960 (Lilly/Novartis), SDZ-268970 (Lilly/Novartis), AM-97 (Armad/Kyowa Hakko), AM-132 (Armad), AM-138 (Armad/Kyowa Hakko), IDN-5005 (Indena), Cryptophycin 52 (also known as LY-355703), AC-7739 (Ajinomoto, also known as AVE-8063A and CS-39.HCl), AC-7700 (Ajinomoto, also known as AVE-8062, AVE-8062A, CS-39-L-Ser.HCl, and RPR-258062A), Vitilevuamide, Tubulysin A, Canadensol, Centaureidin (also known as NSC-106969), T-138067 (Tularik, also known as T-67, TL-138067 and TI-138067), COBRA-1 (Parker Hughes Institute, also known as DDE-261 and WHI-261), H10 (Kansas State University), H16 (Kansas State University), Oncocidin A1 (also known as BTO-956 and DIME), DDE-313 (Parker Hughes Institute), Fijianolide B, Laulimalide, SPA-2 (Parker Hughes Institute), SPA-1 (Parker Hughes Institute, also known as SPIKET-P), 3-IAABU (Cytoskeleton/Mt. Sinai School of Medicine, also known as MF-569), Narcosine (also known as NSC-5366), Nascapine, D-24851 (Asta Medica), A-105972 (Abbott), Hemiasterlin, 3-BAABU (Cytoskeleton/Mt. Sinai School of Medicine, also known as MF-191), TMPN (Arizona State University), Vanadocene acetylacetonate, T-138026 (Tularik), Monsatrol, Inanocine (also known as NSC-698666), 3-1AABE (Cytoskeleton/Mt. Sinai School of Medicine), A-204197 (Abbott), T-607 (Tuiarik, also known as T-900607), RPR-115781 (Aventis), Eleutherobins (such as Desmethyleleutherobin, Desaetyleleutherobin, lsoeleutherobin A, and Z-Eleutherobin), Caribaeoside, Caribaeolin, Halichondrin B, D-64131 (Asta Medica), D-68144 (Asta Medica), Diazonamide A, A-293620 (Abbott), NPI-2350 (Nereus), Taccalonolide A, TUB-245 (Aventis), A-259754 (Abbott), Diozostatin, (−)-Phenylahistin (also known as NSCL-96F037), D-68838 (Asta Medica), D-68836 (Asta Medica), Myoseverin B, D-43411 (Zentaris, also known as D-81862), A-289099 (Abbott), A-318315 (Abbott), HTI-286 (also known as SPA-110, trifluoroacetate salt) (Wyeth), D-82317 (Zentaris), D-82318 (Zentaris), SC-12983 (NCI), Resverastatin phosphate sodium, BPR-OY-007 (National Health Research Institutes), and SSR-250411 (Sanofi).

In some embodiments, a therapeutic agent is an anti-inflammatory agent. In some embodiments, a therapeutic agent is an anti-TNF agent, an IL-1 receptor antagonist, an IL-2 receptor antagonist, a cytotoxic agent, an immunomodulatory agent, an antibiotic, a T-cell co-stimulatory blocker, a B cell depleting agent, an immunosuppressive agent, an alkylating agent, an anti-metabolite, a plant alkaloid, a terpenoids, a topoisomerase inhibitor, an antitumour antibiotic, an antibody, a hormonal therapy, an anti-diabetes agent, a leukotriene inhibitor, or combinations thereof. In some embodiments, a therapeutic agent is selected from: alefacept, efalizumab, methotrexate, acitretin, isotretinoin, hydroxyurea, mycophenolate mofetil, sulfasalazine, 6-Thioguanine, Dovonex, Taclonex, betamethasone, tazarotene, hydroxychloroquine, etanercept, adalimumab, infliximab, abatacept, rituximab, tratuzumab, Anti-CD45 monoclonal antibody AHN-12 (NCI), Iodine-131 Anti-B1 Antibody (Corixa Corp.), anti-CD66 monoclonal antibody BW 250/183 (NCI, Southampton General Hospital), anti-CD45 monoclonal antibody (NCI, Baylor College of Medicine), antibody anti-anb3 integrin (NCI), BIW-8962 (BioWa Inc.), Antibody BC8 (NCI), antibody muJ591 (NCI), indium In 111 monoclonal antibody MN-14 (NCI), yttrium Y 90 monoclonal antibody MN-14 (NCI), F105 Monoclonal Antibody (NIAID), Monoclonal Antibody RAV12 (Raven Biotechnologies), CAT-192 (Human Anti-TGF-Betal Monoclonal Antibody, Genzyme), antibody 3F8 (NCI), 177Lu-J591 (Weill Medical College of Cornell University), TB-403 (Biolnvent International AB), anakinra, azathioprine, cyclophosphamide, cyclosporine A, leflunomide, d-penicillamine, amitriptyline, or nortriptyline, chlorambucil, nitrogen mustard, prasterone, LJP 394 (abetimus sodium), LJP 1082 (La Jolla Pharmaceutical), eculizumab, belibumab, rhuCD40L (NIAID), epratuzumab, sirolimus, tacrolimus, pimecrolimus, thalidomide, antithymocyte globulin-equine (Atgam, Pharmacia Upjohn), antithymocyte globulin-rabbit (Thymoglobulin, Genzyme), Muromonab-CD3 (FDA Office of Orphan Products Development), basiliximab, daclizumab, riluzole, cladribine, natalizumab, interferon beta-1b, interferon beta-1a, tizanidine, baclofen, mesalazine, asacol, pentasa, mesalamine, balsalazide, olsalazine, 6-mercaptopurine, AIN457 (Anti IL-17 Monoclonal Antibody, Novartis), theophylline, D2E7 (a human anti-TNF mAb from Knoll Pharmaceuticals), Mepolizumab (Anti-IL-5 antibody, SB 240563), Canakinumab (Anti-IL-1 Beta Antibody, NIAMS), Anti-IL-2 Receptor Antibody (Daclizumab, NHLBI), CNTO 328 (Anti IL-6 Monoclonal Antibody, Centocor), ACZ885 (fully human anti-interleukin-lbeta monoclonal antibody, Novartis), CNTO 1275 (Fully Human Anti-IL-12 Monoclonal Antibody, Centocor), (3S)-N-hydroxy-4-({(4-[(4-hydroxy-2-butynyl)oxy]phenyl}sulfonyl)-2,2-dimethyl-3-thiomorpholine carboxamide (apratastat), golimumab (CNTO 148), Onercept, BG9924 (Biogen Idec), Certolizumab Pegol (CDP870, UCB Pharma), AZD9056 (AstraZeneca), AZD5069 (AstraZeneca), AZD9668 (AstraZeneca), AZD7928 (AstraZeneca), AZD2914 (AstraZeneca), AZD6067 (AstraZeneca), AZD3342 (AstraZeneca), AZD8309 (AstraZeneca),), [(1R)-3-methyl-1-({(2S)-3-phenyl-2-[(pyrazin-2-ylcarbonyl)amino]propanoyl}amino)butyl]boronic acid (Bortezomib), AMG-714, (Anti-IL 15 Human Monoclonal Antibody, Amgen), ABT-874 (Anti IL-12 monoclonal antibody, Abbott Labs), MRA(Tocilizumab, an Anti IL-6 Receptor Monoclonal Antibody, Chugai Pharmaceutical), CAT-354 (a human anti-interleukin-13 monoclonal antibody, Cambridge Antibody Technology, MedImmune), aspirin, salicylic acid, gentisic acid, choline magnesium salicylate, choline salicylate, choline magnesium salicylate, choline salicylate, magnesium salicylate, sodium salicylate, diflunisal, carprofen, fenoprofen, fenoprofen calcium, flurobiprofen, ibuprofen, ketoprofen, nabutone, ketolorac, ketorolac tromethamine, naproxen, oxaprozin, diclofenac, etodolac, indomethacin, sulindac, tolmetin, meclofenamate, meclofenamate sodium, mefenamic acid, piroxicam, meloxicam, celecoxib, rofecoxib, valdecoxib, parecoxib, etoricoxib, lumiracoxib, CS-502 (Sankyo), JTE-522 (Japan Tobacco Inc.), L-745,337 (Almirall), NS398 (Sigma), betamethasone (Celestone), prednisone (Deltasone), alclometasone, aldosterone, amcinonide, beclometasone, betamethasone, budesonide, ciclesonide, clobetasol, clobetasone, clocortolone, cloprednol, cortisone, cortivazol, deflazacort, deoxycorticosterone, desonide, desoximetasone, desoxycortone, dexamethasone, diflorasone, diflucortolone, difluprednate, fluclorolone, fludrocortisone, fludroxycortide, flumetasone, flunisolide, fluocinolone acetonide, fluocinonide, fluocortin, fluocortolone, fluorometholone, fluperolone, fluprednidene, fluticasone, formocortal, formoterol, halcinonide, halometasone, hydrocortisone, hydrocortisone aceponate, hydrocortisone buteprate, hydrocortisone butyrate, loteprednol, medrysone, meprednisone, methylprednisolone, methylprednisolone aceponate, mometasone furoate, paramethasone, prednicarbate, prednisone, rimexolone, tixocortol, triamcinolone, ulobetasol, Pioglitazone, Rosiglitazone, Glimepiride, Glyburide, Chlorpropamide, Glipizide, Tolbutamide, Tolazamide, Glucophage, Metformin, (glyburide+metformin), Rosiglitazone+metformin, (Rosiglitazone+glimepiride), Exenatide, Insulin, Sitagliptin, (glipizide and metformin), Repaglinide, Acarbose, Nateglinide, Orlistat, cisplatin; carboplatin; oxaliplatin; mechlorethamine; cyclophosphamide; chlorambucil; vincristine; vinblastine; vinorelbine; vindesine; mercaptopurine; fludarabine; pentostatin; cladribine; 5-fluorouracil (5FU); floxuridine (FUDR); cytosine arabino side; trimethoprim; pyrimethamine; pemetrexed; paclitaxel; docetaxel; etopo side; tenipo side; irinotecan; topotecan; amsacrine; etopo side; etopo side phosphate; tenipo side; dactinomycin; doxorubicin; daunorubicin; valrubicine; idarubicine; epirubicin; bleomycin; plicamycin; mitomycin; finasteride; goserelin; aminoglutethimide; anastrozole; letrozole; vorozole; exemestane; 4-androstene-3,6,17-trione (“6-OXO”; 1,4,6-androstatrien-3,17-dione (ATD); formestane; testolactone; fadrozole; A-81834 (3-(3-(1,1-dimethylethylthio-5-(quinoline-2-ylmethoxy)-1-(4-chloromethylphenyl)indole-2-yl)-2,2-dimethylpropionaldehyde oxime-O-2-acetic acid; AME103 (Amira); AME803 (Amira); atreleuton; BAY-x-1005 ((R)-(+)-alpha-cyclopentyl-4-(2-quinolinylmethoxy)-Benzeneacetic acid); CJ-13610 (4-(3-(4-(2-Methyl-imidazol-1-yl)-phenylsulfanyl)-phenyl)-tetrahydro-pyran-4-carboxylic acid amide); DG-031 (DeCode); DG-051 (DeCode); MK886 (1-[(4-chlorophenyl)methyl]3-[(1,1-dimethylethyl)thio]-α,α-dimethyl-5-(1-methylethyl)-1H-indole-2-propanoic acid, sodium salt); MK591 (3-(1-4[(4-chlorophenyl)methyl]-3-[(t-butylthio)-5-((2-quinoly)methoxy)-1H-indole-2]-, dimehtylpropanoic acid); RP64966 ([4-[5-(3-Phenyl-propyl)thiophen-2-yl]butoxy]acetic acid); SA6541 ((R)-S-[[4-(dimethylamino)phenyl]methyl]-N-(3-mercapto-2methyl-1-oxopropyl-L-cycteine); SC-56938 (ethyl-1-[2-[4-(phenylmethyl)phenoxy]ethyl]-4-piperidine-carboxylate); VIA-2291 (Via Pharmaceuticals); WY-47,288 (2-[(1-naphthalenyloxy)methyl]quinoline); zileuton; ZD-2138 (6-((3-fluoro-5-(tetrahydro-4-methoxy-2H-pyran-4yl)phenoxy)methyl)-1-methyl-2(1H)-quinlolinone); doxycycline; or combinations thereof.

Starting Materials

Disclosed herein, in certain embodiments, are molecules of Formula II, having the structure: A₁-X₁-B₁;  Formula II wherein,

-   -   X₁ is a cleavable linker;     -   A₁ is a peptide with a sequence comprising 5 to 9 acidic amino         acids and having a first reactive amino acid moiety c_(A);     -   B₁ is a peptide with a sequence comprising 7 to 9 basic amino         acids and having a second reactive amino acid moiety c_(B); and     -   A₁-X₁-B₁ has a third reactive amino acid moiety c_(M) on A₁ or         X₁; and         wherein c_(A) is capable of reacting with a first cargo moiety         comprising D_(A), c_(B) is capable of reacting with a second         cargo moiety comprising D_(B), and c_(M) is capable of reacting         with a macromolecular carrier comprising M to form a molecule of         Formula I.         In some embodiments, the c_(A), c_(B), and c_(M) have functional         groups that are orthogonally reactive. In some embodiments,         c_(A), c_(B), and c_(M) are each independently selected from a         naturally-occurring amino acid or a non-naturally-occurring         amino acid. In some embodiments, c_(A), c_(B), and c_(M) are         each independently selected from a D amino acid, a L amino acid,         an α-amino acid, a β-amino acid, or a γ-amino acid. In some         embodiments, c_(A), c_(B), and c_(M) are each independently         selected from any amino acid having a free thiol group, any         amino acid having a N-terminal amine group, and any amino acid         with a side chain capable of forming an oxime or hydrazone bond         upon reaction with a hydroxylamine or hydrazine group. In some         embodiments, c_(A), c_(B), and c_(M) are each independently         selected from D-cysteine, D-glutamate, lysine, and para-4-acetyl         L-phenylalanine. In some embodiments, c_(B) is any amino acid         having a free thiol group. In some embodiments, c_(B) is         D-cysteine. In some embodiments, c_(A) is any amino acid having         a N-terminal amine group. In some embodiments, c_(A) is         D-glutamate. In some embodiments, c_(A) is lysine. In some         embodiments, c_(M) is any amino acid with a side chain capable         of forming an oxime or hydrazone bond upon reaction with a         hydroxylamine or hydrazine group. In some embodiments, c_(M) is         para-4-acetyl L-phenylalanine.

As used herein, “orthogonally reactive” means a plurality of groups can be attached to a molecule via a sequence of reactions that do not cross react enabling specific attachment of each group in the presence of the others. In some embodiments, the three groups (D_(A), D_(B), and D_(M)) are able to be attached to A₁-X₁-B₁ via c_(A), c_(B), and c_(M) using a sequence of 3 independent reactions that do not cross react so that each group is attached to only one site on A₁-X₁-B₁.

Disclosed herein, in certain embodiments, is a molecule having the amino acid sequence: (D-Glu)₅-F(4-Ac)-o-Pro-Leu-Gly-Cys(_(Me))-Ala-Gly-(D-Arg)₈-(D-Cys) wherein o represent 5-(amino-3-oxapentanoyl); F_((4-Ac)) represent para-acetyl-(L)-phenylalanine; and C_((Me)) represents S-methyl-(L)-cysteine.

In some embodiments, the molecule further comprises a polyethylene glycol (PEG) polymer. In some embodiments, the PEG polymer is covalently linked to the molecule at the F(4-Ac) subunit. In some embodiments, the molecule comprises groups that can be orthogonally reacted. In some embodiments, the groups that can be orthogonally reacted are chosen from: an amine, thiol and an acetyl phenylalanine. In some embodiments, the molecule comprises an amine, a thiol, and an acetyl phenylalanine.

In some embodiments, the PEG polymer has an average molecular weight of 500 daltons. In some embodiments, the PEG polymer has an average molecular weight of 2,000 daltons. In some embodiments, the PEG polymer has an average molecular weight of 5,000 daltons. In some embodiments, the PEG polymer has an average molecular weight of 10,000 daltons. In some embodiments, the PEG polymer has an average molecular weight of 20,000 daltons. In some embodiments, the PEG polymer has an average molecular weight of 40,000 daltons. Disclosed herein, in certain embodiments, is the use of the molecule in the synthesis of a molecule according to Formula I.

Disclosed herein, in certain embodiments, is a molecule having the amino acid sequence: (D-Glu)₅-o-Pro-Leu-Glys-Cys_((me))-Ala-Gly-(D-Arg)₈-(D-Cys)-[PEG_((3K))] wherein all glutamates and arginines are D-amino acids; o represents 5-(amino-3-oxapentanoyl); C(me) represents S-methyl-(L)-cysteine; and PEG(_(3K)) represents α-amino-ω-amide poly(ethylene glycol) with an average three thousand Dalton molecular weight. In some embodiments, the molecule further comprises a fluorescent moiety. Disclosed herein, in certain embodiments, is the use of the molecule in the synthesis of a molecule according to Formula I.

EXAMPLES Materials and Methods

HPLC-grade acetonitrile was purchased from Fisher Scientific (Phillipsburg, Pa.). Purified water was collected through Milli-Q water purification system (Millipore, Bedford, Mass.). 3-Maleimidopropionic acid-Pfp ester was purchased from Molecular Biosciences (Boulder, Colo.). PBS-EDTA buffer was purchased from Teknova (Hollister, Calif.). Trifluoroacetic acid (TFA), Dimethylformamide (DMF) and N-methylmorpholine (NMM) were supplied by Sigma-Aldrich (Milwaukee, Wis.). α-Mercaptoethyl-ω-methoxy, poly-oxyethylene (Mw 2,000, 5,000, 20,000 and 40,000) [mPEG(2K)-SH, mPEG(5K)-SH, mPEG(20K)-SH, mPEG(40K)-SH] and α-aminoxyl-ω-methoxy, polyoxyethylene (Mw 2,000, 5,000, 20,000 and 40,000) [mPEG(2K)-ONH₂, mPEG(5K)-ONH₂, mPEG(20K)-ONH₂, mPEG(40K)-ONH₂] were purchased from NOF America Corporation (Irvine, Calif.). mPEG(1K)-NHNH₂ was purchased from Nanocs (New York). IRDye 800CW maleimide (Mal-IRDye) and IRDye 750 succinimidyl ester were supplied by Li-Cor Biosciences (Lincoln, Nebr.). Lyophilized peptides P1-P17 were prepared using standard resin based peptide coupling methods.

LC-MS analysis was carried out on an Agilent 1200 SL series in combination with AB SCIEX API 3200, equipped with CTC PAL autosampler operating at 4° C., a vacuum degasser, binary pump, UV-VIS detector, associated Analyst 1.5 analytical software and a Phenomenex column (Kinetex 2.6μ C18 100 Å, 100×2.1 mm) or a Waters 2695 separation module equipped with a Waters 2487 dual λ absorbance detector in combination with Finnigan LCQ Deca XP mass spectrometer. The equipment is associated with Xcalibur analytical software and a Peeke Scientific column (Titan 200 5 μm, C18-MC, 50×2.1 mm).

Preparation HPLC were carried out on an Agilent system (Agilent 1200 series) and a Thermo Scientific column (Hypersil Gold C18, 5μ, 250×10 mm), or a Waters Delta Prep preparative HPLC System and a Varian column (F75L, C18, 15μ, 1200 g), or a Waters PrepLC System equipped with a Waters 2487 dual λ absorbance detector, Fraction Collector III, Masslynx software and a Thermo Scientific column (Hypersil Gold C18, 5μ, 250×10 mm) or a Phenomenex column (luna, C18(2), 5μ, 100 Å AX 150×30 mm). The mobile phase consisted of a water (0.05% TFA)(solvent A)/acetonitrile (0.05% TFA)(solvent B) gradient.

Centrifugation was carried out at 4° C. with an Eppendorf centrifuge 5810R or a Beckman Microfuge® 18.

Exemplary materials for synthesis of the selective delivery molecules disclosed herein include, but are not limited to, any of peptides P-1, P-2, P-3, P-4, P-5, P-6, P-7, P-8, P-9, P-10, P-11, P12, P-13, P-14, P-15, P-16, and P-17.

The above starting materials are summarized below.

Peptide Sequences Peptide P-1 eeeeeeeeeoPLGC_((Me))AGrrrrrrrrrc Peptide P-2 eeeeeoPLGC_((Me))AGrrrrrrrrc Peptide P-3 eeeeeF_((4-Ac))oPLGC_((Me))AGrrrrrrrrc Peptide P-4 eeeeeeeeeF_((4-Ac))oPLGC_((Me))AGrrrrrrrrrc Peptide P-5 (Ac)eeeeeoPLGC_((Me))AGrrrrrrrrck Peptide P-6 eeeeeoPLGC_((Me))AGoF_((4-Ac))rrrrrrrrc Peptide P-7 eeeeeeeeeoPLGC_((Me))AGrrrrrrrrrcoF_((4-Ac)) Peptide P-8 [mPEG_((2K))]crrrrrrrrPLGC_((Me))AGoeeeeek Peptide P-9 [mPEG_((5K))]crrrrrrrrPLGC_((Me))AGoeeeeek Peptide P-10 eeeeeoPLGC_((Me))AGrrrrrrrrc[PEG_((3K))] Abbreviations: Standard I letter amino acid abbreviations were used in all the sequences. Lowercase characters indicated D-amino acids. All peptides were amidated at C-terminus. o: 5-(amino-3-oxapentanoyl); F_((4-Ac)): para-acetyl-(L)-phenylalanine; C(_(Me)): S-methyl-(L)-cysteine. PEG(3K): α-amino-ω-amide poly(ethylene glycol) with an averaged three thousand Daltons molecular weight; mPEG(2k): α-carboxy-ω-methoxy poly(ethylene glycol) with an averaged two thousand Daltons molecular weight; mPEG(5k): α-carboxy-ω-methoxy poly(ethylene glycol) with an averaged five thousand Daltons molecular weight. Ac: acetyl.

Example 1 Synthesis of SDM-2 from Peptide P-1

Synthesis of Intermediate 5

To a solution of peptide P-1 (8 mg, 2.1 μmol) in DMF (0.8 mL) at room temperature in the dark were added IRDye 800CW maleimide (2 mg, 1.7 μmol) and N-methylmorpholine (10 μL, 91 μmol) with stirring. The reaction was followed by LC-MS and usually completed in 1 h. The mixture was directly used in the next step without further purification.

To the reaction mixture above was added 3-maleimidopropionic acid-Pfp ester (2 mg, 6.0 μmol). The resulting mixture was stirred at room temperature in the dark for 20 h. Purification by RP-HPLC afforded intermediate 5 (2.1 mg, 22% for two steps). Calculated: [M+3H]³⁺ (C₁₈₇H₂₉₀N₅₉O₆₄S₆) m/z=1526. Found ESI: [M+3H]³⁺ (C₁₈₇H₂₉₀N₅₉O₆₄S₆) m/z=1526.

Synthesis of Selective Delivery Molecule SDM-2

The mixture of intermediate 5 (1.5 mg, 0.27 μmol) and mPEG(40K)-SH (10 mg, 0.25 μmol) in PBS-EDTA buffer (0.5 mL, 137 mM NaCl, 7 mM Na₂HPO₄, 3 mM KCl, 1.4 mM K₃PO₄, 4 mM EDTA, pH 7.4) was stirred at room temperature in the dark for 20 h. Purification by RP-HPLC afforded selective delivery molecule SDM-2 (7.0 mg, 61%).

Selective delivery molecules SDM-1, SDM-3, SDM-4, and SDM-5 were prepared analogously to SDM-2 from peptide P-1.

Example 2 Synthesis of SDM-6 from Peptide P-2

Synthesis of Intermediate 7

To a solution of peptide P-2 (378.5 mg, 0.1 mmol) in DMF (25 mL) at room temperature in the dark were added Cy5 maleimide (87 mg, 0.09 mmol) and N-methylmorpholine (350 μL, 3.2 mmol) with stirring. The reaction was followed by LC-MS and completed in 1 h. The mixture was directly used in the next step without further purification.

To the reaction mixture above was added 3-maleimidopropionic acid-Pfp ester (50 mg, 0.15 mmol). The resulting mixture was stirred at room temperature in the dark for 5 h. Purification by RP-HPLC afforded intermediate 7 (108 mg, 27% for two steps). Calculated: [M+2H]²⁺ (C₁₄₈H₂₃₅N₅₁O₄₄S₄) m/z=1780. Found ESI: [M+2H]²⁺ (C₁₄₈H₂₃₅N₅₁O₄₄S₄) m/z=1780.

Synthesis of Selective Delivery Molecule SDM-6

The mixture of intermediate 7 (95 mg, 21.2 μmol) and mPEG(40K)-SH (0.9 g, 22.5 μmol) in PBS-EDTA buffer (40 mL, 137 mM NaCl, 7 mM Na₂HPO₄, 3 mM KCl, 1.4 mM K₃PO₄, 4 mM EDTA, pH 7.4) was stirred at room temperature in the dark for 20 h. Purification by RP-HPLC afforded selective delivery molecule SDM-6 (0.85 g, 90%).

Selective delivery molecules SDM-7 and SDM-8 were prepared analogously to SDM-6 from peptide P-2.

Example 3 Synthesis of SDM-25 from Peptide P-3

Synthesis of Intermediate 8

To a solution of peptide P-3 (200 mg, 49.6 μmol) in DMF (5 mL) at room temperature in the dark were added Cy5 maleimide (60 mg, 65.6 μmol) and N-methylmorpholine (80 μL, 0.73 mmol) with stirring. The reaction was followed by LC-MS and completed in 1 h. Ether (40 mL) was added to the mixture. The precipitate was collected after centrifuge, washed with ether (40 mL×2) and purified by HPLC to afford intermediate 8 (141 mg, 61%). Calculated: [M+3H]³⁺ (C₁₅₂H₂₄₂N₅₁O₄₃S₄) m/z=1200. Found ESI: [M+3H]³⁺ (C₁₅₂H₂₄₂N₅₁O₄₃S₄) m/z=1200.

Synthesis of Intermediate 9

To a solution of intermediate 8 (101 mg, 21.8 μmol) in DMF (10 mL) at room temperature were added Cy7 carboxylic acid, succinimidyl ester (40 mg, 41.1 μmol) and N-methylmorpholine (0.2 mL, 1.8 mmol). The resulting mixture was stirred at room temperature in the dark for 36 h. Ether (35 mL) was added to the mixture. The precipitate was collected after centrifuge and washed with ether (40 mL×2). Purification of the mixture by RP-HPLC afforded intermediate 9 (28.1 mg, 25%) and intermediate 8 (63 mg). Calculated: [M+3H]³⁺ (C₁₈₇H₂₈₂N₅₃O₅₀S₆) m/z=1421. Found ESI: [M+3H]³⁺ (C₁₈₇H₂₈₂N₅₃O₅₀S₆) m/z=1421.

Synthesis of Selective Delivery Molecule SDM-25

The mixture of intermediate 9 (28.1 mg, 5.4 μmol) and mPEG(2K)-ONH₂ (17 mg, 7.6 μmol) in glycine buffer (4 mL, 0.1 M, 20 mM aniline, pH 3.0) and acetonitrile (0.8 mL) was stirred at room temperature in the dark for 24 h. After the reaction was complete, acetophenone (10 μL, 86 μmol) was added. The mixture was stirred at room temperature for 2 h. Purification by RP-HPLC afforded selective delivery molecule SDM-25 (25 mg, 63%).

Selective delivery molecules SDM-9, SDM-10, SDM-22, SDM-23, SDM-24, SDM-26, SDM-27, SDM-29 and SDM-31 were prepared analogously to SDM-25 from peptide P-3.

Example 4 Synthesis of SDM-15 from Peptide P-4

Synthesis of Intermediate 11

To a solution of peptide P-4 (30 mg, 6.2 μmol) in DMF (2 mL) at room temperature in the dark were added Cy5 maleimide (7.5 mg, 8.2 μmol) and N-methylmorpholine (15 μL, 0.14 mmol) with stirring. The reaction was followed by LC-MS and completed in 1 h. The mixture was purified by HPLC to afford intermediate 11 (19.7 mg, 59%). Calculated: [M+3H]³⁺ (C₁₇₈H₂₈₂N₅₉O₅₆S₄) m/z=1424. Found ESI: [M+3H]³⁺ (C₁₇₈H₂₈₂N₅₉O₅₆S₄) m/z=1424.

Synthesis of Intermediate 12

To a solution of intermediate 11 (15 mg, 2.8 μmol) in DMF (1.5 mL) at room temperature were added Cy7 carboxylic acid, succinimidyl ester (4 mg, 4.3 μmol) and N-methylmorpholine (10 μL, 91 μmol). The resulting mixture above was stirred at room temperature in the dark for 48 h. Purification by RP-HPLC afforded intermediate 12 (5.0 mg, 30%). Calculated: [M+3H]³⁺ (C₂₁₃H₃₂₂N₆₁O₆₃S₆) m/z=1645. Found ESI: [M+3H]³⁺ (C₂₁₃H₃₂₂N₆₁O₆₃S₆) m/z=1645.

Synthesis of Selective Delivery Molecule SDM-15

The mixture of intermediate 12 (1.1 mg, 0.18 μmol) and mPEG(2K)-ONH₂ (1 mg, 0.5 μmol) in glycine buffer (1 mL, 0.1 M, 20 mM aniline, pH 3.0) and acetonitrile (0.2 mL) was stirred at room temperature in the dark for 1 day. Purification by RP-HPLC afforded selective delivery molecule SDM-15 (0.6 mg, 42%).

Selective delivery molecules SDM-11, SDM-12, SDM-13, SDM-14 and SDM-28 were prepared analogously to SDM-15 from intermediate 11.

Example 5 Synthesis of SDM-16 from Peptide P-5

Synthesis of Intermediate 20

To a solution of peptide P-5 (20 mg, 5.2 μmol) in DMF (1 mL) at room temperature in the dark were added Cy5 maleimide (6 mg, 6.6 μmol) and N-methylmorpholine (12 μL, 109 μmol) with stirring. The reaction was followed by LC-MS and usually completed in 1 h. The mixture was directly used for the next step without further purifications. To a solution of the above mixture in DMF (1 mL) at room temperature was added 3-maleimidopropionic acid-Pfp ester (2.5 mg, 7.5 μmol). The resulting mixture above was stirred at room temperature in the dark for 20 h. Purification by RP-HPLC afforded intermediate 20 (7.3 mg, 30% for two steps). Calculated: [M+3H]³⁺ (C₁₅₆H₂₅₀N₅₃O₄₆S₄) m/z=1244. Found ESI: [M+3H]³⁺ (C₁₅₆H₂₅₀N₅₃O₄₆S₄) m/z=1244.

Synthesis of Selective Delivery Molecule SDM-16

The mixture of intermediate 20 (1.4 mg, 0.3 μmol) and mPEG(40K)-SH (14 mg, 0.35 μmol) in PBS-EDTA buffer (2 mL, 137 mM NaCl, 7 mM Na₂HPO₄, 3 mM KCl, 1.4 mM K₃PO₄, 4 mM EDTA, pH 7.4) was stirred at room temperature in the dark for 20 h. Purification by RP-HPLC afforded selective delivery molecule SDM-16 (6.5 mg, 49%).

Selective delivery molecules SDM-17, SDM-18 were prepared analogously to SDM-16 from peptide P-5.

Example 6 Synthesis of SDM-33 from Peptide P-7

Synthesis of Intermediate 21

To a solution of peptide P-7 (20 mg, 4.1 μmol) in DMF (1 mL) at room temperature in the dark were added Cy5 maleimide (6 mg, 6.6 μmol) and N-methylmorpholine (10 μL, 91 μmol) with stirring. The reaction was followed by LC-MS and completed in 1 h. The mixture was purified by RP-HPLC to afford intermediate 21 (9 mg, 40%). Calculated: [M+3H]³⁺ (C₁₈₂H₂₈₉N₆₀O₅₈S₄) m/z=1458. Found ESI: [M+3H]³⁺ (C₁₈₂H₂₈₉N₆₀O₅₈S₄) m/z=1458.

Synthesis of Intermediate 22

To a solution of intermediate 21 (9 mg, 1.6 μmol) in DMF (1 mL) at room temperature were added Cy7 carboxylic acid, succinimidyl ester (3 mg, 3.1 μmol) and N-methylmorpholine (10 μL, 91 μmol). The resulting mixture above was stirred at room temperature in the dark for 24 h. Purification by RP-HPLC afforded intermediate 22 (4.9 mg, 50%). Calculated: [M+3H]³⁺ (C₂₁₇H₃₂₉N₆₂O₆₅S₆) m/z=1679. Found ESI: [M+3H]³⁺ (C₂₁₇H₃₂₉N₆₂O₆₅S₆) m/z=1679.

Synthesis of Selective Delivery Molecule SDM-33

The mixture of intermediate 22 (0.9 mg, 0.15 μmol) and mPEG(10K)-ONH₂ (3 mg, 0.3 μmol) in glycine buffer (1 mL, 0.1 M, 20 mM aniline, pH 3.0) and acetonitrile (0.2 mL) was stirred at room temperature in the dark for 3 days. After the reaction was complete, acetophenone (10 μL, 86 μmol) was added. The mixture was stirred at room temperature for 2 h. Purification by RP-HPLC afforded selective delivery molecule SDM-33 (0.8 mg, 38%).

Selective delivery molecule SDM-34 was prepared analogously to SDM-33 from intermediate 22.

Example 7 Synthesis of SDM-36 from Peptide P-8

Synthesis of Intermediate 23

To a solution of peptide P-8 (10 mg, 1.7 μmol) in DMF (1 mL) at room temperature in the dark were added Cy5 maleimide (4 mg, 4.4 μmol) and N-methylmorpholine (10 μL, 91 μmol) with stirring. The reaction mixture was stirred at room temperature in 1 h. Purification by RP-HPLC afforded intermediate 23 (5.4 mg, 48%).

Synthesis of Selective Delivery Molecule SDM-36

To a solution of intermediate 23 (5.4 mg, 0.82 μmol) in DMF (1 mL) at room temperature were added Cy7 carboxylic acid, succinimidyl ester (3 mg, 3.1 μmol) and N-methylmorpholine (10 μL, 91 μmol). The resulting mixture above was stirred at room temperature in the dark for 36 h. Purification by RP-HPLC afforded SDM-36 (0.7 mg, 13%).

Selective delivery molecules SDM-37 was prepared analogously to SDM-36 from peptide P-8.

Example 8 Synthesis of SDM-38 from Peptide P-10

Synthesis of Intermediate 24

To a solution of peptide P-10 (10 mg, 1.4 μmol) in PBS buffer (pH 7.4, 1 mL) at room temperature in the dark were added Cy5 maleimide (4 mg, 4.4 μmol) with stirring. The reaction mixture was stirred at room temperature in 1 h. Purification by RP-HPLC afforded intermediate 24 (7.9 mg, 79%).

Synthesis of Selective Delivery Molecule SDM-38

To a solution of intermediate 24 (7.9 mg, 1.1 μmol) in DMF (1 mL) at room temperature were added Cy7 carboxylic acid, succinimidyl ester (2 mg, 2.0 μmol) and N-methylmorpholine (10 μL, 91 μmol). The resulting mixture above was stirred at room temperature in the dark for 36 h. Purification by RP-HPLC afforded selective delivery molecules SDM-38 (1.7 mg, 19%).

Selective delivery molecules SDM-39 was prepared analogously to SDM-38 from peptide P-10.

Example 9 Synthesis of SDM-40 from Peptide P-8

Synthesis of Intermediate 25

To a solution of peptide P-8 (10 mg, 1.7 μmol) in DMF (1 mL) at room temperature in the dark were added Cy7 maleimide (4 mg, 4.2 μmol) and N-methylmorpholine (10 μL, 91 μmol) with stirring. The reaction mixture was stirred at room temperature in 1 h. Purification by RP-HPLC afforded intermediate 23 (3.1 mg, 28%).

Synthesis of Selective Delivery Molecule SDM-40

To a solution of intermediate 23 (3.1 mg, 0.47 μmol) in DMF (1 mL) at room temperature were added Cy5 carboxylic acid, succinimidyl ester (2 mg, 2.1 μmol) and N-methylmorpholine (5 μL, 46 μmol). The resulting mixture above was stirred at room temperature in the dark for 24 h. Purification by RP-HPLC afforded SDM-40 (1.4 mg, 41%).

Example 10 Synthesis of SDM-30 from Peptide P-3

Synthesis of Intermediate 26

The mixture of intermediate 8 (3 mg, 0.64 μmol) and Cy7-ONH₂ (3 mg, 2.9 μmol) in glycine buffer (4 mL, 0.1 M, 20 mM aniline, pH 3.0) and acetonitrile (0.1 mL) was stirred at room temperature in the dark for 36 h. Purification by RP-HPLC afforded intermediate 26 (1.1 mg, 31%). Calculated: [M+3H]³⁺ (C₁₈₉H₂₈₈N₅₅O₅₀S₆) m/z=1441. Found ESI: [M+3H]³⁺ (C₁₈₉H₂₈₈N₅₅O₅₀S₆) m/z=1441. Cy7-ONH₂ was prepared from Cy7-COOH and 2-[N-phthalimido-(aminooxy)]ethanamine under standard amide coupling conditions followed by the removal of the phthalimide protecting group with hydrazine. 2-[N-phthalimido-(aminooxy)]ethanamine was prepared from commercially available N-Boc-ethanolamine and N-hydroxyphthalimide through a Mitsunobu reaction followed by the cleavage of Boc group with TFA.

Synthesis of Intermediate 27

To a solution of intermediate 26 (1.1 mg, 0.2 μmol) in DMF (1 mL) at room temperature were added 3-maleimidopropionic acid-Pfp ester (0.5 mg, 1.5 μmol) and N-methylmorpholine (5 μL, 45 μmol). The resulting mixture above was stirred at room temperature in the dark for 36 h. Purification by RP-HPLC afforded intermediate 27 (0.8 mg, 75%). Calculated: [M+3H]³⁺ (C₁₉₆H₂₉₁N₅₆O₅₃S₆) m/z=1491. Found ESI: [M+3H]³⁺ (C₁₉₆H₂₉₁N₅₆O₅₃S₆) m/z=1491.

Synthesis of Selective Delivery Molecule SDM-30

The mixture of intermediate 27 (0.7 mg, 0.15 μmol) and mPEG(10K)-SH (3 mg, 0.3 μmol) in PBS-EDTA buffer (0.5 mL, 137 mM NaCl, 7 mM Na₂HPO₄, 3 mM KCl, 1.4 mM K₃PO₄, 4 mM EDTA, pH 7.4) was stirred at room temperature in the dark for 40 h. Purification by RP-HPLC afforded selective delivery molecule SDM-30 (1.2 mg, 23%).

Selective delivery molecules SDM-32 and SDM-35 were prepared analogously to SDM-30 from peptides P-3 and P-4.

Example 11 Synthesis of SDM-21 from Peptide P-6

Synthesis of Intermediate 28

To a solution of peptide P-6 (30 mg, 7.6 μmol) in DMF (2 mL) at room temperature in the dark were added Cy5 maleimide (9 mg, 9.4 μmol) and N-methylmorpholine (15 μL, 137 μmol) with stirring. The reaction was followed by LC-MS and completed in 1 h. Purification by RP-HPLC afforded intermediate 28 (24.9 mg, 68%). Calculated: [M+3H]³⁺ (C₁₅₆H₂₄₉N₅₂O₄₅S₄) m/z=1233. Found ESI: [M+3H]³⁺ (C₁₅₆H₂₄₉N₅₂O₄₅S₄) m/z=1233.

Synthesis of Intermediate 29

To a solution of intermediate 28 (17.7 mg, 3.7 μmol) in DMF (1.5 mL) at room temperature were added Cy7 carboxylic acid, succinimidyl ester (5 mg, 5.5 μmol) and N-methylmorpholine (20 μL, 0.18 mmol). The resulting mixture was stirred at room temperature in the dark for 30 h. Purification of the mixture by RP-HPLC afforded intermediate 29 (7.1 mg, 35%). Calculated: [M+3H]³⁺ (C₁₉₁H₂₈₉N₅₄O₅₂S₆) m/z=1455. Found ESI: [M+3H]³⁺ (C₁₉₁H₂₈₉N₅₄O₅₂S₆) m/z=1455.

Synthesis of Selective Delivery Molecule SDM-21

The mixture of intermediate 29 (1.8 mg, 0.33 μmol) and mPEG(10K)-ONH₂ (4 mg, 0.4 μmol) in glycine buffer (1 mL, 0.1 M, 20 mM aniline, pH 3.0) and acetonitrile (0.1 mL) was stirred at room temperature in the dark for 3 days. Purification by RP-HPLC afforded selective delivery molecule SDM-21 (1.0 mg, 20%).

Selective delivery molecules SDM-19 and SDM-20 were prepared analogously to SDM-21 from intermediate 29.

Example 12 Enzyme Dependent Fluorescence Enhancement and Color Changes

Selective delivery molecule 9 was dissolved in TCNB buffer (50 mM Tris, pH 7.5, with 10 mM calcium chloride, 150 mM sodium chloride, and 0.05% BRIJ 35) at room temperature at 1 μM. Fluorescence spectra were recorded on F-2500 fluorescence spectrometer. The Cy5 fluorescence donor was excited using 625 nm light and the emission was scanned from 660 to 800 nm. The Cy5 donor emission peaked at −670 nm and the Cy7 FRET acceptor emission peak was ˜780 nm as shown in FIG. 2. Peptide cleavage was initiated with addition of matrix metalloproteinase-2 (MMP-2) at a final concentration of 1 nM. The cleavage reaction was complete within 2 hour and the fluorescence spectra indicated FRET disruption and a large 8-fold increase the Cy5 donor emission and 2-fold decrease in the Cy7 emission. The actual intrinsic fluorescence decrease of Cy7 is larger however it is masked by the Cy5 long wavelength shoulder. This result demonstrates that SDM-9 has efficient energy transfer from Cy5 to Cy7 in the intact peptide.

Example 13 Enzyme Dependent Fluorescence Enhancement and Color Changes

Selective delivery molecule 10 was dissolved in TCNB buffer (pH 7.5) at room temperature at 1 μM. Fluorescence spectra were recorded on F-2500 fluorescence spectrometer. Excitation of the Cy5 fluorescence donor was excited at 625 nm and the emission was measured at 669 nm. Peptide cleavage was initiated with addition of MMP-9 at a final concentration of 1 nM. The cleavage reaction was complete within 2 hour and the fluorescence was enhanced >100-fold upon protease cleavage, FIG. 3. The large fluorescence response demonstrates that the dye quencher efficiently quenches the Cy5 fluorophore in the uncleaved SDM-10.

Example 14 Fluorogenic Response from Tumor Homogenates

HT1080 cells (Cat. # CCL-121; American Type Culture Collection, VA, USA) were grown under exponential growth conditions in humidified atmosphere of 5% CO₂ in air at 37° C. until reaching 80-100% confluence before harvesting for mouse implantation. Each nude mouse was hand restrained and injected with 2×10⁶ HT-1080 cells into the mammary fat pad using a 25-G needle. HT-1080 tumors were harvested when they had reached 100-200 mm³ in size (typically 1-2 weeks post-tumor cells implantation).

HT-1080 tumors were homogenized using ultrasonic disruption. 1 nM MMP-9 or 10 μL tumor tissue homogenates (TH2 and TH3) were mixed with 1 μM SDM-10 in 100 μL buffer for 24 h at 37° C. Selective delivery molecule 6 was used as a fluorescent control of similar size to intact SDM-10. The samples were loaded on a polyacrylamide gel and separated using electrophoresis. The data are shown in FIG. 4 and demonstrate that SDM-10 is essentially non-fluorescent prior to cleavage. After incubation with HT-1080 tumor homogenates, SDM-10 is cleaved and becomes highly fluorescent. GM6001 is a general broad spectrum inhibitor of MMPs. The fact that GM6001 inhibits cleavage demonstrates that the homogenate cleavage is due to tumor associated MMPs.

Example 15 In Vivo Imaging Assay for Tumor Contrast

HT-1080 xenograft model was generated as described in Example 14 and used to evaluate the ability of molecules to provide in vivo tumor fluorescence contrast compared to surrounding tissue. Fluorescent conjugates were tested in HT-1080 tumor-bearing mice once the tumors had reached 100-200 mm³ in size (typically 1-2 weeks post-tumor cells implantation). Conscious HT-1080 tumor-bearing mice were restrained using a rotating tail injector (Cat. #RTI; Braintree Scientific, MA, USA) and dosed intravenously (tail vein) with the test compound at between 0.1 and 5 nanomoles per mouse in 100 uL saline solution. In preparation for imaging, mice were lightly anesthetized with a mixture of ketamine/xylazine (Cat. #K-113; Sigma, Aldrich, Mo., USA) given intraperitoneally (1 μL/gram body weight) to minimize movement.

Serial whole-body imaging (tumor included) was done using a whole-animal fluorescent visualization imaging system or Olympus stereo fluorescent microscope. The mice were positioned on their backs and imaging was performed from the top to image the ventral side of the animal. Excitation and emission wavelengths were selected based on the fluorescent dye used. Contrast was calculated using the following equation: Contrast=(Fluorescence intensity of tumor−Fluorescence intensity of contralateral chest tissue)/Intensity of contralateral chest tissue).

Contrast greater than 0.4 in the whole animal is easily detected by eye in the whole animal image and is good contrast. Contrast >0.7 is high contrast.

The mice were imaged several times between 1-24 hours after injection.

Representative imaging data two hours after dosing for selective delivery molecule 6 in 3 different mice is shown in FIG. 1. In this particular image the mean contrast is 1.1. Other compounds were tested in a similar fashion and the contrast results are given in table 1.

TABLE 1 Summary of peptide conjugate in vivo contrast data from HT-1080 xenograft model. Maximum Contrast Time to maximum contrast (hr) Selective delivery (≧0.4 = good; (very fast <4; 4 < fast < 12; molecule ≧0.9 = high) >12 slow) 1 Good Fast 2 High Fast 3 High Slow 4 5 6 High Very Fast 7 High Slow 8 Good Very fast 9 High Fast

Example 16 In Vivo Distribution and Compounds with Improved Tissue Accumulation

To determine the total dye accumulation in various organs, HT-1080 xenograft mice were sacrificed and tissue samples from blood, liver, kidney, and tumor were collected 6 hours after compounds were administered iv via the tail vein. 3-4 mice were used for each data point. Blood samples were stored at 4° C. overnight and then centrifuged at 15,000 rpm to separate out the serum. The organs were mixed in a ProK buffer (0.25 mg/ml Prok, 0.1 mg/ml DNAse, 150 mM NaCl, 10 mM Tris pH8.0, 0.2% SDS) at 10 μL/mg tissue and cut into small pieces using scissors. The tissue/digest solution was then sonicated for 1 minute at 67% duty cycle and digested overnight at 37° C. After digestion, the sample was centrifuged at 15,000 rpm and the tissue homogenate was aspirated off and stored at 4° C.

The tissue concentration of fluorescent compounds were determined from fluorescence standard curves generated by spiking in know concentrations of administered compounds into serum and tissue homogenates (at various dilutions) from control animals that were not injected with compound. The linear range for each compound was determined for each tissue. Fluorescence measurements were done on either a fluorescent plate reader or fluorescence spectrometer. The tissue biodistribution results from selective delivery molecules 1, 2, and 6 are shown in FIG. 5. A surprising result was that selective delivery molecule 6 has 5-fold higher tissue distribution into tumor compared to selective delivery molecules 1 and 2. This unexpected result is due to the asymmetric core composed of uneven numbers of positively and negatively charged peptide backbone. Selective delivery molecules 1 and 2 have equal numbers giving a net neutral core while selective delivery molecule 6 has a net 3+ charge due to more positively charged arginines. This demonstrates that compounds with different number of acidic and basic amino acids have improved and useful in vivo and biodistribution properties over symmetric molecules.

Example 17 In Vivo Detection of Cancer Metastases to Lymph Node with FRET SDMs Fluorescence Labeling of Metastatic Cervical Lymph Nodes Following Intravenous and Peritumoral Administration of Fluorescent SDMs in Tumor Bearing Mice

The following model and assays were used to determine the ability of fluorescent SDMs to detect cancer metastases to lymph nodes in immunocomptent BALB/c mice (Charles River, Wilmington, Mass. 01887) bearing syngeneic ear tumors.

Mouse Model.

The mice were housed in groups of 4 in individually ventilated IVC disposable cages (Innovive, Inc., San Diego, Calif. 92121) and had free access to standard laboratory chow (Cat. #2018, Harlan Laboratories, Inc. Indianapolis, Ind. 46250) and drinking water. Animals were kept under controlled environmental conditions (12-h/12-h light/dark cycle) for at least 5 days before tumor cell implantation. All experimental procedures were carried out under the approved IACUC protocol # EB11-002-009A. Murine 4T1 tumor (ATCC® Number: CRL-2539™) and mammary carcinoma (Polyoma Middle T 8119 subclone “PyMT 8119”) cells from the American Type Culture Collection (ATCC, Manassas, Va. 20108) and the University of San Diego, Calif. (UCSD, La Jolla, Calif. 92093) respectively were grown separately using standard cell culture techniques. Tumor cells (4×10⁵ tumor cells/50 μL/mouse) were suspended in DPBS/Matrigel™ (1:1 vol) and injected subcutaneously on the mouse ear pinna above the auricular cartilage for primary tumor induction. The in vivo imaging of metastatic cervical lymph nodes in ear tumor-bearing mice used as surrogate murine model of metastatic breast cancer took place seventeen to twenty days following tumor cell implantation.

Test SDM Compound Administration.

For the intravenous administration (tail vein injection) of SDMs, mice were restrained in a rotating tail injector (Cat. #RTI, Braintree Scientific, Inc., Braintree, Mass. 02185) and the test article (5-120 μM; 100 μL/mouse) injected in mouse using a 28G^(1/2) insulin syringe (Cat. #14-826-79, Becton Dickinson and Company, Franklin Lakes, N.J. 07417). To perform the peritumoral injection of SDMs, each involved mouse was sedated using the ketamine/xylazine (Ketaject® & Xyla-ject®, Phoenix Pharmaceuticals, St. Joseph, Mo. 64506) mixture administered intraperitoneally and the test article (5-120 μM; 30-60 μL/ear) injected subcutaneously around the primary tumor and contralateral ear pinna using a 30 G PrecisionGlide™ needle (Cat. #305106, Becton Dickinson and Company, Franklin Lakes, N.J. 07417). After dosing, each mouse was returned to the assigned cage and kept under controlled environmental conditions before imaging. Fluorescence imaging of cervical lymph nodes 1-24 hours after compound administration as described below.

Fluorescence Imaging.

To image the cervical lymph nodes, each mouse was deeply anesthetized with a mixture of ketamine/xylazine administered intraperitoneally. The deeply anesthetized mouse was transferred on a piece of black cork (4×4 inches, Quartet®, ACCO Brands, Lincolnshire, Ill. 60069, USA) for blunt dissection and imaging of cervical lymph nodes using a computerized fluorescent stereomicroscope (SZX10, Olympus Optical, CO, LTD, Japan) equipped with appropriate fluorescence filters for both single intensity and two fluorophore fluorescence ratio detection. For example, filters for Cy5 and Cy7 were used for FRET-based SDMs with Cy5 and Cy7 FRET pair. After in vivo fluorescence imaging (see below for ratio imaging method), the cervical lymph nodes were surgically removed, fixed in 10% buffered formalin and processed for histology (Hematoxylin & Eosin staining) to assess the fluorescence/cancer correlation and determine diagnostic performance of SDMs.

Emission Ratio Imaging Method.

Fluorescence images were acquired using an Olympus SZX10 Research Stereo Microscope (Olympus America, Center Valley, Pa.). For Cy5 and Cy7 FRET-based SDMs an excitation filter centered at 620 nm (Chroma ET620/60x, Chroma Technology Corp. Bellows Falls, Vt.) and emission filters centered at 700 nm and 810 nm (Chroma filters ET700/75m and ET810/90m) were used to produce two images at different emission wavelengths. Images were acquired with an Orca-R2 camera (Hamamatsu, Bridgewater, N.J.) connected to a Windows-based computer. Two methods were used to determine emission ratios for lymph nodes. For one method the intensity was averaged over a region of interest (ROI) drawn to include part or all of the lymph node of interest. The Emission ratio was then calculated from the intensity data for each region of interest. Roi EmissionRatio=(roiInt1/Exp1)/(Int2/Exp2)  (equation 1) where

roiInt1=averaged intensity for ROI at emission wavelength 1 with ET700/75m filter

Exp1=exposure time used for Int1

roiInt 2=average intensity for ROI at emission wavelength 2 with ET810/90m filter

Exp 2=exposure time used for Int2

A second method used to determine emission ratios was based averaging the emission ratio from a region of interest (ROI) drawn to include part or all of the lymph node of interest taken from an emission ratio image. Emission ratio images were produced by using a modified form of equation 1 that included a scaling factor so that the pixel values would fall between 0 and 255 for an 8-bit image. Px EmissionRatio=k*(pxInt1/Exp1)/(pxInt2/Exp2)  (equation 2)

where

k=scaling factor

pxInt1=pixel intensity at emission wavelength 1 with ET700/75m filter

Exp1=exposure time used for Int1

pxInt 2=pixel intensity at emission wavelength 2 with ET810/90m filter

Exp 2=exposure time used for Int2

Emission ratios for lymph nodes gave quantitatively similar results using either method.

Lymph nodes were identified as either metastatic or non-metastatic by a pathologist based on H&E staining. Emission ratio contrast for each SDM (selective delivery molecule) was then quantified by dividing the average emission ratio of the metastatic nodes by the average emission of the non-metastatic nodes and subtracting one as shown in equation 3: ERC=MetAV/ConAV−1  (Equation 3) where

ERC=emission ratio contrast

MetAV=average metastatic lymph node emission ratio

ConAV=average non-metastatic contralateral lymph node emission ratio

An example of an emission ratio image is shown in FIG. 6. The right hand panel show the ratio image which show high contrast between the metastatic lymph node (very large node indicated with lower left dark arrow) and the non-metastatic nodes (other arrows). The higher ratio is shown as lighter pixels (metastatic) compared to darker lower ratio pixels for the non-metastic nodes.

Useful for detecting cancerous lymph nodes, a contrast of 20 to 50% was considered good, an increase of 50 to 100% was considered high, while an increase greater than 100% was considered to be very high contrast.

TABLE 2 Summary of SDMs in vivo ratio contrast data from Murine 4T1 tumor model. IV Maximum Contrast Peritumor Maximum Contrast Selective (Low <20%, Good 20% to (Low <20%, Good 20% delivery 50%, High >50% to 100% to 50%, High >50% to 100%, molecule Very High >100%) Very High >100%) SDM-9 nd Low SDM-11 nd Low SDM-12 nd Good SDM-13 Good Good SDM-14 Good Very High SDM-19 Good SDM-20 Low SDM-21 nd Good SDM-22 Low SDM-23 High Very High SDM-24 Very High Very High SDM-25 Very High Very High SDM-27 Very High nd SDM-28 Good High SDM-29 nd High SDM-30 nd Very High SDM-31 Low nd SDM-32 Very High Very High SDM-33 Low High SDM-35 Very High Good SDM-36 High Good SDM-37 Low nd SDM-38 Good nd SDM-39 Good High SDM-40 nd High

Example 18 Ex Vivo Mouse PyMT 8119 Tumor Activity Assay: SDM Cleavage and FRET Emission Ratio Response in Mouse Cancer Tissue Compared to Non Cancerous Tissue

Tumor and muscle tissue samples from PyMT 8119 tumor bearing mice were collected and frozen at −80° C. The tissues were thawed and homogenized in cold TCNB buffer (pH 7.5, 50 mM Tris-HCl, 10 mM CaCl₂, 150 mM NaCl and 0.05% Brij35) at 100 mg/200 μL using ultrasonic disruption (VCX500, Sonics & Materials Inc, Newtown, Conn.). After homogenates were centrifuged at 15,000 g at 4° C. for 20 min, supernatants were collected. APMA (p-aminophenylmercuric acetate, 90 μL, 2 mM in TCNB buffer) was added to the supernatants (90 μL). The resulting mixtures were incubated at 37° C. for 1 h before use. 500 nM of SDM-23 was used for the cleavage of 45 μL of activated tissue supernatants (final volume: 50 μL). The assay was carried out using a SpectraMax M2 spectrometer with SoftMax Pro v4.5 software. Fluorescence signals of (λex, 620 nm, λem, 670 nm), (λex, 620 nm, λem, 773 nm) and (λex, 720 nm; λem, 773 nm), where λex and λem stand for excitation and emission wavelengths respectively, were measured as a function of time at room temperature. Samples were measured in triplicate and the FRET SDM cleavage resulted in an increased Cy5/Cy7 fluorescence emission ratio where Cy5 signal used (λex, 620 nm, λem, 670 nm) and Cy7 620 nm, λem, 773 nm) experimental conditions.

Enzymatic activity from the tissues resulted in SDM-23 cleavage and generated a large FRET emission ratio increase (labeled primary tumor), as shown in FIG. 7. The ratio increase is the result of SDM cleavage. These data show that SDM-23 is very active in mouse breast cancer tissues and cleavage is significantly greater in cancerous tissue compared to normal muscle, which shows not activity in this assay.

Example 19 Human Ex Vivo Tissue Assay: SDM Cleavage and FRET Emission Ratio Response in Human Cancer Tissue Compared to Noncancerous Tissue

Human breast cancer tissue samples and normal human breast tissue (provided by Cancer Human Tissue Network) were homogenized in cold TCNB buffer (pH 7.5, 50 mM Tris-HCl, 10 mM CaCl₂, 150 mM NaCl and 0.05% Brij35) at 100 mg/200 μL using ultrasonic disruption (VCX500, Sonics & Materials Inc, Newtown, Conn.). After homogenates were centrifuged at 15,000 g at 4° C. for 20 min, supernatants were collected. 500 nM of SDM was used for the cleavage of 45 uL of tissue supernatant (final volume: 50 μL) in the assay unless otherwise noted. The assay was carried out using a SpectraMax M2 spectrometer with SoftMax Pro v4.5 software. Fluorescence signals of (λex, 620 nm, λem, 670 nm), (λex, 620 nm, 773 nm) and (λex, 720 nm; λem, 773 nm), where λex and λem stand for excitation and emission wavelengths respectively, were measured as a function of time at room temperature. Samples were measured in triplicate and the FRET SDM cleavage resulted in an increased Cy5/Cy7 fluorescence emission ratio where Cy5 signal used (λex, 620 nm, λem, 670 nm) and Cy7 (λex, 620 nm, λem, 773 nm) experimental conditions. An example using SDM-25 is shown in FIG. 8. Other SDMs were evaluated using the same procedure. The cleavage dependent fluorescence response can also be quantified as the rate of cleavage (delta ratio per time), as shown in FIG. 9 for SDM-25 and SDM-32. The rates were calculated from the slope of the data from time 0 to 300 minutes.

Example 20 High Diagnostic Sensitivity and Specificity for an SDM in a Metastatic Lymph Node Model

Key performance metrics of a diagnostic agent are sensitivity and specificity. Sensitivity relates to the ability to correctly diagnose test positives. While specificity relates to the ability to correctly diagnose test negatives.

As an example of high diagnostic performance of a FRET SDM, we use data generated from SDM-24 in the 4T1 mouse metastatic lymph model. SDM-24 was administered via IV tail vein injection. After 3 to 6 hours, the mice lymph nodes were imaged using fluorescence ratio imaging as described previously to determine whether or not the lymph node had a high ratio (diagnosed cancer positive) or low ratio (diagnosed cancer negative). Sensitivity and specificity was determined using receiver operating characteristic (ROC) or ROC curves (http://en.wikipedia.org/wiki/Receiver_operating_characteristic). For ROC curve analysis, data is divided into a binary classification of positives and negatives based on a threshold value for the emission ratio. The ROC curve plots true positive fraction of positives (true positive rate) versus false positive fraction of negatives (false positive rate).

True positives, false positives, true negatives, and false negatives were determined by comparing the prediction based on the fluorescence emission ratio data and threshold value with the positive or negative assignment made by a pathologist using H&E staining. The emission ratio values for the cancer positive and negatives (as determined by H&E histopathology) are shown in FIG. 10. The threshold value was gradually adjusted from low to high to obtain a full ROC curve from (1, 1) or all positives to (0, 0) or all negatives. A ROC curve is shown in FIG. 11. Data from 48 lymph nodes were used to generate this curve. Note that sensitivity and specificity can be determined for each point in the ROC curve. Sensitivity is the true positive rate while specificity is one minus the false positive rate. Equations used to generate the ROC curve are shown below. TPR=TP/(TP+FN) FPR=FP/(FP+TN)

Where:

TPR=true positive rate

FPR=false positive rate

TP=# of true positives

TN=# of true negatives

FP=# of false positives

FN=# of false negatives

In this example both sensitivity and specificity are 100% for all threshold values between 5.65 and 7.15. This means that all lymph nodes were correctly identified with the FRET emission ratio method when compared to the gold standard histopathology. Generally, sensitivity and specificity values >90% are considered very high.

Example 21 Use of an SDM to to Visualize Cancer in Breast Cancer Patients

SDM-25 is delivered intravenously to a breast cancer patient. The fluorescent moieties on SDM-25 are taken up by cancerous cells and/or tissue after cleavage of the linker. A light source is shined onto the target tissue. The fluorescent moieties emit light which is detected by a camera or a detector. The data obtained by the camera or detector is processed to generate an image that allows the surgeon to visualize cancerous cells or tissue. The surgeon excises said tissue for biopsy.

Example 22 Use of an SDM to to Visualize Cancer in Prostate Cancer Patients

SDM-26 is delivered intravenously to a prostate cancer patient. The fluorescent moieties on SDM-26 are taken up by cancerous cells and/or tissue after cleavage of the linker. A light source is shined onto the target tissue. The fluorescent moieties emit light which is detected by a camera or a detector. The data obtained by the camera or detector is processed to generate an image that allows the surgeon to visualize cancerous cells or tissue. The surgeon excises said tissue for biopsy.

Example 23 Use of an SDM to Visualize Cancer in Patients with Head and Neck (Squamous) Cancer

SDM-27 is delivered intravenously to a head and neck cancer patient. The fluorescent moieties on SDM-27 are taken up by cancerous cells and/or tissue after cleavage of the linker. A light source is shined onto the target tissue. The fluorescent moieties emit light which is detected by a camera or a detector. The data obtained by the camera or detector is processed to generate an image that allows the surgeon to visualize cancerous cells or tissue. The surgeon excises said tissue for biopsy.

Example 24 Use of an SDM to Visualize Cancer in Patients with Melanoma

SDM-24 is delivered intravenously to a patient having melanoma. The fluorescent moieties on SDM-24 are taken up by cancerous cells and/or tissue after cleavage of the linker. A light source is shined onto the target tissue. The fluorescent moieties emit light which is detected by a camera or a detector. The data obtained by the camera or detector is processed to generate an image that allows the surgeon to visualize cancerous cells or tissue. The surgeon excises said tissue for biopsy.

Example 25 Use of an SDM to Visualize Cancer in Patients with Thyroid Cancer

SDM-32 is delivered intravenously to a thyroid cancer patient. The fluorescent moieties on SDM-32 are taken up by cancerous cells and/or tissue after cleavage of the linker. A light source is shined onto the target tissue. The fluorescent moieties emit light which is detected by a camera or a detector. The data obtained by the camera or detector is processed to generate an image that allows the surgeon to visualize cancerous cells or tissue. The surgeon excises said tissue for biopsy. 

What is claimed is:
 1. A selective delivery molecule of Formula I, having the structure: [D_(A)-c _(A)]-A-[c _(M)-M]-X-B-[c _(B)-D_(B)]  Formula I wherein, X is a peptide linker cleavable by a matrix metalloproteinase; A is a peptide with a sequence comprising a series of 5 glutamates (SEQ ID NO:5); B is a peptide with a sequence comprising a series of 8 arginines (SEQ ID NO:6); c_(A), c_(B), and c_(M) are each independently an amino acid; M is a polyethylene glycol (PEG) polymer; and D_(A) and D_(B) are a pair of acceptor and donor fluorescent moieties that are capable of undergoing Försters/fluorescence resonance energy transfer with the other; and wherein [c_(M)-M] is bound to at any position on X, [D_(A)-c_(A)] is bound to any amino acid on A, [c_(B)-D_(B)] is bound to any amino acid on B, and M is a PEG polymer having an average molecular weight of 500 Da to 10 kDa.
 2. The molecule of claim 1, wherein c_(A), c_(B), and c_(M) are each independently selected from D-cysteine, D-glutamate, lysine, and para-4-acetyl L-phenylalanine.
 3. The molecule of claim 2, wherein c_(B) is D-cysteine.
 4. The molecule of claim 2, wherein c_(A) is D-glutamate.
 5. The molecule of claim 2, wherein c_(A) is lysine.
 6. The molecule of claim 2, wherein c_(M) is para-4-acetyl L-phenylalanine.
 7. The molecule of claim 1, wherein X comprises an amino acid sequence that is cleavable by MMP2, MMP7, MMP9, or MMP14.
 8. The molecule of claim 7, wherein X comprises an amino acid sequence selected from: PLGLAG (SEQ ID NO:2), PLG-C(me)-AG (SEQ ID NO:1), RPLALWRS (SEQ ID NO:7), ESPAYYTA (SEQ ID NO:8), DPRSFL (SEQ ID NO:9), PPRSFL (SEQ ID NO:10), RLQLKL (SEQ ID NO:11), and RLQLK(Ac) (SEQ ID NO:12).
 9. The molecule of claim 1, wherein D_(A) and D_(B) are Cy5 and Cy7.
 10. The molecule of claim 1, wherein D_(A) and D_(B) are Cy5 and IRDye750.
 11. The molecule of claim 1, wherein D_(A) and D_(B) are Cy5 and IRDye800.
 12. The molecule of claim 1, wherein D_(A) and D_(B) are Cy5 and ICG.
 13. The molecule of claim 1, wherein the molecule of Formula I is: i) SDM-14 ii) SDM-15 iii) SDM-23 iv) SDM-24 v) SDM-25 vi) SDM-26 vii) SDM-27 viii) SDM-32; or ix) SDM-35.
 14. The molecule of claim 1, wherein M is a PEG polymer having an average molecular weight of 500 Da.
 15. The molecule of claim 1, wherein M is a PEG polymer having an average molecular weight of 2 kDa.
 16. The molecule of claim 1, wherein M is a PEG polymer having an average molecular weight of 5 kDa.
 17. The molecule of claim 1, wherein M is a PEG polymer having an average molecular weight of 10 kDa.
 18. The molecule of claim 7, wherein X comprises an amino acid sequence selected from: PLGLAG (SEQ ID NO:2), PLG-C(me)-AG (SEQ ID NO:1), and RPLALWRS (SEQ ID NO:7).
 19. The molecule of claim 7, wherein X comprises the amino acid sequence PLG-C(me)-AG (SEQ ID NO:1). 